Optimized microbial cells for production of melatonin and other compounds

ABSTRACT

Described herein are recombinant microbial host cells comprising biosynthetic pathways and their use in producing oxidation products and downstream products, e.g., melatonin and related compounds, as well as enzyme variants, nucleic acids, vectors and methods useful for preparing and using such cells. In specific aspects, the present invention relates to monooxygenases, e.g., amino acid hydroxylases, with a modified cofactor-dependency, and to enzyme variants and microbial cells providing for an improved supply of cofactors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This U.S. non-provisional patent application is a national phaseapplication of PCT/EP2017/057520, entitled “Optimized Microbial Cellsfor Production of Melatonin and Other Compounds,” filed Mar. 30, 2017,which itself claims priority to European patent application numberEP16170405.1 entitled “Optimized Microbial Cells for Production ofMelatonin and Other Compounds,” filed May 19, 2016, and to U.S.provisional application No. 62/315,865, entitled “Optimized MicrobialCells for Production of Melatonin and Other Compounds,” filed Mar. 31,2016, the contents of which are all fully incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to recombinant microbial host cellscomprising biosynthetic pathways and their use in producing oxidationproducts of aromatic amino acids, such as, e.g., 5HTP, as well asdownstream products such as melatonin and related compounds. The presentinvention also relates to enzymes and enzyme variants and their use insuch recombinant microbal cells, as well as methods and vectors usefulfor preparing and selecting such cells. More specifically, the presentinvention relates to microbial host cells repurposed for optimalmonooxygenase, e.g., amino acid hydroxylase, function, and to methods ofpreparing and using such cells.

BACKGROUND OF THE INVENTION

Melatonin is a hormone secreted by the pineal gland in the brain which,inter alia, maintains the body's circadian rhythm, is involvedregulating other hormones, and is a powerful anti-oxidant. Because of,e.g., its role in regulating circadian rhythm, melatonin has beenavailable for many years as an over-the-counter dietary supplement inthe U.S. This melatonin is, however, typically chemically synthesized,and there is a need for a simplified and more cost-effective procedure.

In animals, melatonin is biosynthesized from the native metaboliteL-tryptophan via the intermediates 5-hydroxy-L-tryptophan (5HTP),serotonin and N-acetylserotonin. The first step is this pathway, theconversion of L-tryptophan to 5HTP, is catalyzed by L-tryptophanhydroxylase (TPH). TPH and many other mammalian aromatic amino acidhydroxylases require oxygen and tetrahydropterin (BH4) as cofactors.

WO 2013/127914 A1, WO 2013/127915 A1 and WO 2015/032911 A1 (DanmarksTekniske Universitet) describe the production of 5HTP or melatonin inrecombinant E. coli and S. cerevisiae cells expressing a TPH and enzymesof a biosynthetic pathway for producing the BH4 co-factor. BH4 does notoccur naturally in E. coli or S. cerevisiae but can be biosynthesizedfrom endogenous GTP via a pathway comprising a GTP cyclohydrolase I(GCH1), a 6-pyruvoyl-tetrahydropterin synthase (PTS or PTPS) and asepiapterin reductase (SRP) (Yamamoto et al., 2003; Ehrenworth et al.,2015), and regenerated into BH4 via consecutive reactions bypterin-4α-carbolamine dehydratase (PCD) and dihydropterin reductase(DHPR) (e.g., WO 2015/032911 and Ehrenworth et al., 2015). Nar et al.(1995) and Rebelo et al. (2003) have reported on the structure of the E.coli GCH1; FolE. The amino acid sequence of FolE is reported inUniProtKB P0A6T5 (SEQ ID NO:16). An alternative FolE sequence isprovided by NCBI Reference Sequence WP_001639660.

US 2014/134689 AA (University of California) describes methods ofproducing oxidation products such as L-DOPA, 5HTP, serotonin and/ormelatonin from aromatic amino acids in a host cell which can, e.g., becapable of biosynthesizing BH4 or tetrahydromonapterin (MH4) from GTP.MH4 was reportedly capable of replacing THB as cofactor for tyrosinehydroxylase (TH).

MH4 is endogenous to, e.g., E. coli and P. aeruginosa cells, where it ishypothesized to be the cofactor for phenylalanine hydroxylase (PheH),its formation requiring FolX and FolM (Pribat et al., 2010a).Phenylalanine hydroxylases from non-flowering plants, however, uniquelypreferred 10-formyltetrahydrofolate (10-THF) as cofactor over BH4 andwere not capable of using MH4 (Pribat et al., 2010b).

Lin et al. (2014) describes that E. coli transformed with engineeredbacterial phenylalanine 4-hydroxylases and a “regeneration” pathwaybased on a PCD and endogenous folM (dihydromonapterin reductase; DHMR)could convert tryptophan to 5HTP using endogenous MH4 as a co-factor.Hara and Kino (2013) produced 5HTP in a similar E. coli system,reportedly increasing the yield with the addition of BH4 cofactor.

Despite these and other advances in the art, there is still a need forrecombinant microorganisms capable of efficient production of melatoninand related compounds. It is an object of the invention to provide suchmicroorganisms.

SUMMARY OF THE INVENTION

It has been found by the present inventor that, surprisingly, oxidationproducts of amino acids, such as 5HTP, can be produced in a recombinantmicrobial cell comprising a heterologous monooxygenase, such as a TPH,and a heterologous PCD. Optionally, the microbial cell further comprisesother enzymes for converting the oxidation product into a desiredend-product. For example, the recombinant microbial cell may compriseheterologous enzymes converting 5HTP into serotonin and/or melatonin. Insome embodiments, this is possible even in the absence of exogenousnucleic acid encoding a DHPR or a DHMR. While not being limited totheory, this suggests that native E. coli compounds may support themonooxygenase (TPH) activity.

So, in one aspect the present invention relates to a recombinantmicrobial cell comprising heterologous nucleic acid sequences encoding amonoxygenase, such as a TPH, and a PCD, wherein the microbial celloptionally does not comprise an exogenous nucleic acid encoding a DHPRor a DHMR, and to the use of such a recombinant cell for producing anoxidation product of an amino acid, e.g., an aromatic amino acid.

The present inventor has also identified variants of GCH1 which improvemonooxygenase activity in recombinant microbial cells comprising amonooxygenase and a PCD, as well as variants of TPH with improvedhydroxylation activity.

So, in other aspects, the present invention relates to such variantenzymes, as well as to nucleic acids and vectors encoding such variants,to recombinant microbial cells expressing such variants, and to the useof such enzymes and recombinant microbial cells for producing oxidationproducts, typically according to the preceding aspect.

To provide for production of other compounds of interests, therecombinant microbial cell can further comprise nucleic acids encodingother enzymes. For example, to produce compounds such as serotonin,N-acetyl-serotonin and melatonin, the recombinant microbial cell maycomprise heterologous nucleic acid sequences encoding a 5HTPdecarboxylase (ADDC), a serotonin acetyltransferase (AANAT) and/or anacetylserotonin O-methyltransferase (ASMT), as described further below.

In further aspects, the invention relates to methods of producing 5HTP,L-DOPA or tyrosine, as well as downstream products such as, e.g.,serotonin, melatonin and hydroxytyrosol, using such recombinantmicrobial cells.

In addition, the present inventor has identified a nucleic acid sequencecomprising a Trc promoter and a DNA coding sequence encoding an enzymeor other protein, wherein the 3′-end of the Trc promoter is operably,preferably directly, linked to the 5′-end of the DNA coding sequence,and wherein the 5′-end sequence of the DNA coding sequence is atgaaa.

So, in other aspects, the present invention relates to such nucleic acidsequences, their use in expressing enzymes or other proteins, to methodsof improving expression of an enzyme or other protein in an E. coli hostcell, and to variant enzymes or other proteins so produced.

These and other aspects and embodiments are described in more detailbelow.

LEGENDS TO THE FIGURES

FIG. 1: An overview of pterin and folic acids biosynthesis from GTP inE. coli. Black solid arrows indicate native E. coli biosynthesispathways of folic acids, tetrahydromonapterin (H₄-MPt), and preQ₀ from acommon precursor, dihydroneopterin triphosphate (H₂—NPtP₃). Theheterologous tetrahydrobiopterin (H₄—BPt) biosynthesis and recyclingpathways are also shown. Arrow in dashed line shows a putative reaction.Thin arrows represent formation of 5-formyl-tetrahydrofolate (5-CHO—FH₄)in a side reaction of GlyA and recycling via Ygfa. The insert showsselected pterin and folate compounds in E. coli which share a commonhead group with H₄—BPt. Genes are labeled in italics. Otherabbreviations: aromatic amino acid hydroxylase (AAH),dihydroneopterin-3-phosphate (H₂—N₃P), dihydromonapterin triphosphate(H₂-MPtP₃), 6-pyruvoyl-tetrahydrobiopterin (H₄—PPt),6-carboxy-tetrahydropterin (H₄—CPt), quinonoid dihydrobiopterin(q-H₂—BPt), 4α-OH—H₄—BPt (pterin-4α-carbinolamine), dihydropteroate(H₂—Pte), dihydrofolate (H₂-folate), tetrahydrofolate (H₄-folate),5-methyl-tetrahydrofolate (5-CH₃—FH₄), 5,10-methenyltetrahydrofolate(5,10-CH₂—FH₄), 5,10-methylenetetrahydrofolate (5,10-CH—FH₄),10-formyl-tetrahydrofolate (10-CHO—FH₄), and p-aminobenzoate (pAB).

FIG. 2: FolE(T198I) mutation cannot be compensated by overexpression.TpH-dependent cell growth were evaluated under four conditions. 1) asingle-copy native chromosomal FolE (gFolE), 2) a single-copy nativechromosomal FolE and a native copy expressed from plasmid (gFolE+pFolE),3) a single-copy native chromosomal FolE and a mutant copy expressedfrom plasmid (gFolE+pFolE*) and 4) a single-copy mutant chromosomal FolE(gFolE*) FIG. 3: ClustalW alignment of ocTPH (SEQ ID NO:1), hsTPH1 (SEQID NO:2), mmTPH (SEQ ID NO:7), ggTPH (SEQ ID NO:6), hsTPH2 (SEQ IDNO:3), btTPH (SEQ ID NO:), ssTPH (SEQ ID NO:5), ecTPH (SEQ ID NO:8) andscTPH (SEQ ID NO:5) indicating, in underlined text, the residuescorresponding to residues E147, L148, N242 and P244 in Homo sapiens TPH2(SEQ ID NO:3).

FIG. 4: Alignment of GCH1 from E. coli (“E coli,” SEQ ID NO:16), S.cerevisiae (“yeast”; SEQ ID NO:17) and Homo sapiens (“human”, SEQ IDNO:14), indicating the residues corresponding to residues D97-E112,K121-D130, N170-H180, S193-L200 and S207-N222 in E. coli GCH1 (SEQ IDNO:16).

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a recombinant microbial cell comprisingnucleic acid sequences, optionally heterologous nucleic acid sequences,encoding a monooxygenase, a PCD and a GCH1. As shown by the results inExample 1, surprisingly, it is possible to use native bacterialTHB-resembling species to support heterologous, optionally mammalian,TPH activity, with functional expression of a heterologous PCD genebeing the only requirement. Additionally, by laboratory evolution, itwas further possible to identify specific mutations in, e.g., GCH1providing for a 10-fold increase in 5HTP production as compared to theparent E. coli cell.

In one aspect, the invention provides a variant of E. coli GTPcyclohydrolase I (FolE) having at least about 80% sequence identity tonative E. coli GCH1 (SEQ ID NO:16) and comprising one or more mutations,wherein, in an E. coli cell comprising a pterin-4α-carbinolaminedehydratase (PCD) and at least one of a tryptophan hydroxylase (TPH), atyrosine hydroxylase (TH) and a phenylalanine hydroxylase (PheH), thevariant provides for an increased hydroxylation activity of at least oneof the TPH, TH and PheH as compared to native E. coli GCH1, and themutation is not T198P. In one embodiment, at least one of the one ormore mutations is in an amino acid residue is in a segment selected fromD97-E112, K121-D130, N170-H180, S193-L200 and S207-N222. For example, atleast one of the one or more mutations can be in an amino acid residueselected from the group consisting of D97, M99, T101, V102, A125, K129,N170, V179, T196, T198, S199, L200, S207, H212, E213, F214, L215 andH221. Non-limiting examples according to this aspect are those whereinthe variant

-   -   (a) comprises a mutation selected from D97V, D97L, D97A, D97T,        M99C, M99T, M99V, M99L, M99I, T101I, T101V, T101L, V102M, N170K,        N170D, N170L, V179A, V179M, T196I, T196V, T196L, T198I, T198V,        T198S, T198L, S199Y, S199F, L200P, L200C, L200S, L200A, S207R,        S207K, S207M, H212R, H212K, E213K, E213R, F214A, F214G, F214S,        L215P, L215Q, L215N, L215D, L215T, L215S, L215G, L215A, L215C,        L215F, L215M, H221R and H221K, or a combination thereof;    -   (b) has at least about 90% sequence identity to native E. coli        GCH1;    -   (c) provides for a hydroxylation activity of at least about        120%, such as at least 130%, as compared to native E. coli GCH1;        or    -   (d) a combination of any two or more of (a) to (d).

Preferably, the GCH1 variant of any such aspect or embodiment comprisesa mutation selected from T198I, T198S, F214S, V179A, M99I, L200P andL215P. The mutation may, for example, be T198I.

In another aspect, the invention provides a variant of a TPH, comprisinga segment corresponding to residues E147 to T460 of Homo sapiens TPH(SEQ ID NO:3), an N-terminal methionine residue, and a mutation in atleast one of the residues corresponding to residues E147, N242 and P244in SEQ ID NO:3, optionally wherein the variant is a variant of a Homosapiens, Schistosoma mansoni, Gallus gallus, Sus scrofa, Mus musculus orOryctolagus cuniculus TPH, optionally wherein the segment in the varianthas at least about 80% sequence identity to the segment in the nativesequence. In separate and specific embodiments, the mutation in theresidue corresponding to residue E147 is an amino acid substitutionselected from 147K, 147R and 147H; the mutation in the residuecorresponding to residue N242 is an amino acid substitution which is242I; and/or the mutation in the residue corresponding to P244 isselected from 244C, 244D, 244L and 244Q, e.g., 244 C or 244D, such as244D.

In another aspect, the invention provides a nucleic acid sequenceencoding such variant E. coli GCH1, Homo sapiens TPH, or both,optionally in the form of one or more vectors which may further compriseone or more expression control sequences. The coding sequence of avariant Homo sapiens TPH may, for example, have the 5′-end atgaaa and beoperably linked to a Trc promoter.

In another aspect, the invention provides a recombinant microbial cellcomprising a variant E. coli GTP GCH1 as described herein or a nucleicacid sequence encoding such a variant. The recombinant microbial cellmay further comprise nucleic acid sequences, optionally heterologous,encoding a monooxygenase and a PCD. In preferred embodiments, therecombinant microbial cell is one wherein

-   -   (a) the PCD is selected from Chromobacterium violecum, Homo        sapiens, Pseudomonas aeruginosa and Rattus norvegicus; or a        functionally active variant, homolog or fragment thereof;    -   (b) the monooxygenase is a TPH selected from a Schistosoma        mansoni, Homo sapiens, Gallus gallus, Bos taurus, Sus scrofa,        Equus caballus, Mus musculus and Oryctolagus cuniculus TPH; or a        functionally active variant, homolog or fragment of any thereof;    -   (c) the monooxygenase is a TH selected from Rattus norwegicus,        Homo sapiens, Mus musculus, Bos taurus, Gallus gallus or a        functionally active variant, homolog or fragment thereof;    -   (d) the monooxygenase is a PheH selected from Chromobacterium        violaceum, Xanthomonas campestris pv. Viticola, Pseudomonas        aeruginosa, Pseudomonas putida, Homo sapiens, Mus musculus,        Streptomyces coeruleorubidus or a functionally active variant,        homolog or fragment thereof; or    -   (e) a combination of (a) and (b), (a) and (c) or (a) and (d).

In some embodiments, each nucleic acid sequence is operably linked to aninducible, a regulated or a constitutive promoter, and/or at least onenucleic acid encoding an enzyme (e.g., the GCH1 variant), ischromosomally integrated.

In some embodiments, the recombinant microbial cell further comprises

-   -   (a) a nucleic acid sequence encoding a 5HTP decarboxylase        (ADDC);    -   (b) nucleic acid sequences encoding an ADDC and a serotonin        acetyltransferase (AANAT);    -   (c) nucleic acid sequences encoding an ADDC, an AANAT, and an        acetylserotonin O-methyltransferase (ASMT); and/or    -   (d) nucleic acid sequences encoding a dopa decarboxylase, a        tyramine oxidase and an alcohol dehydrogenase.

The invention also provides a method of producing one or more oxidationproducts of an aromatic amino acid, comprising culturing the recombinantmicrobial cell of any aspect or embodiment in a medium comprising acarbon source, and, optionally, isolating the oxidation product. Theoxidation product may, for example, comprise at least one of 5HTP,L-DOPA, tyrosine, m-tyrosine, serotonin, melatonin and hydroxytyrosol.

In another aspect, the invention provides a method of selecting and,optionally, producing, a variant GCH1, the method comprising the stepsof

-   -   (a) preparing a population of variants of a parent GCH1;    -   (b) preparing a population of microbial cells, each expressing a        variant GCH1, a PCD, and at least one hydroxylase selected from        a TPH, a TH, and a PheH;    -   (c) analyzing the population of microbial cells for        hydroxylation of a substrate for the hydroxylase as compared to        a control, optionally wherein the control is a microbial cell        expressing parent GCH1, the PCD and the at least one        hydroxylase;    -   (d) selecting at least one variant GCH1 which provides increased        hydroxylation as compared to the control; and    -   (e) optionally, producing the variant GCH1 by recombinant        expression in a host cell.

In another aspect, the invention provides a nucleic acid sequencecomprising a Trc promoter and a DNA coding sequence encoding a protein,wherein the 3′-end of the Trc promoter is directly linked to the 5′-endof the DNA coding sequence, and wherein the 5′-end sequence of the DNAcoding sequence is atgaaa.

In a related aspect, the invention provides a method of producing arecombinant variant of a parent protein in an E. coli host cell, whereinthe N-terminal amino acid sequence of the parent enzyme or other parentprotein is MX (Met-Xaa), wherein X is not K (Lys), the method comprisingthe steps of:

-   -   (a) preparing a vector comprising a Trc promoter directly linked        to a DNA coding sequence encoding a variant of the parent        protein which has an X2K amino acid substitution or an insertion        of a K between the N-terminal M and X, wherein the 5′-end        sequence of the DNA coding sequence is atgaaa;    -   (b) transforming an E. coli host cell with the vector of (a);    -   (c) expressing the recombinant variant of the parent protein in        the host cell; and, optionally, harvesting the recombinant        variant of the parent protein.

Definitions

Unless otherwise specified or contradicted by context, amino acidresidue numbers in Homo sapiens TPH2 herein refer to their position inthe sequence provided by NCBI accession No. NP_775489 and UniprotKBreference Q8IWU9 (SEQ ID NO:3).

Unless otherwise specified or contradicted by context, amino acidresidue numbers in E. coli GCH1 (FolE) herein refer to their position inthe sequence provided by UniprotKB reference P0A6T5 (SEQ ID NO:16).

As used herein, “exogenous” means that the referenced item, such as amolecule, activity or pathway, is added to or introduced into the hostcell or microorganism. An exogenous nucleic acid sequence can, forexample, be introduced either as chromosomal genetic material byintegration into a host chromosome or as non-chromosomal geneticmaterial such as a plasmid. Such an exogenous nucleic acid sequence canencode an enzyme or enzyme activity which is either heterologous to thehost cell or organism in question or which is an endogenous enzyme orenzyme activity in the host cell or organism. Likewise, an exogenousmolecule such as a substrate or cofactor can be added to or introducedinto the host cell or microorganism, e.g., via adding the molecule tothe media in or on which the host cell or microorganism resides.

In the present context the term “heterologous” means that the referenceditem, such as a molecule, activity or pathway, does not normally appearin the host cell or microorganism species in question. Typically, aheterologous pathway comprises at least one nucleic acid sequence,enzyme or other component which is heterologous to the host cell.

As used herein, the terms “native” or “endogenous” mean that thereferenced item is normally present in or native to the host cell ormicrobial species in question.

As used herein, “upregulating” an endogenous gene means increasing thetranscription and/or translation of a gene present in the native hostcell genome relative to a control, such as e.g. the unmodified hostcell. Methods of upregulating genes are known in the art and include,e.g., introducing a non-native promoter increasing transcription,modifying the native promoter, deleting genes encoding repressorprotein, introducing multiple copies of the gene of interest, etc.“Downregulating” an endogenous gene as used herein means to reduce,optionally eliminate, the transcription or translation of an endogenousgene relative to a control, such as, e.g., the unmodified host cell.Methods of down-regulating, disrupting and deleting genes are known tothose of skill in the art, and include, e.g., site-directed mutagenesis,genomic modifications based on homologous recombination, RNA degradationbased on CAS9, etc.

In the present context, “overexpressing” refers to introducing anexogenous nucleic acid sequence encoding an enzyme which is eitherheterologous or native to the microbial host cell, or is a functionally(i.e., catalytically) active fragment or variant thereof, and expressingthe exogenous nucleic acid sequence to increase the enzyme activity inthe microbial cell as compared to the microbial host cell without theintroduced exogenous nucleic acid sequence, e.g., a native microbialhost cell. This can be useful if, e.g., a microbial host cell does notnormally contain the enzymatic activity referred to, where the nativeenzymatic activity is insufficient, or the native enzyme is subjected tounwanted regulation. In such cases, an exogenous nucleic acid sequenceencoding an enzyme which is heterologous to the microbial host cell andwhich has the desired activity and regulation patterns can beintroduced. Overexpression of a nucleic acid sequence can be achieved byplacing the nucleic acid sequence under the control of a strongpromoter. Non-limiting examples of strong promoters suitable for, e.g.,E. coli cells are Ptrc, Plac, PlacUV5, PT7, and PTrp. Non-limitingexamples of strong promoters suitable for, e.g., yeast cells are TEF1,PGK1, HXT7 and TDH3.

As used herein, a gene that is a “homolog” or “homologous” to anothergene is generally an ortholog (i.e., a descended from the same ancestralsequence but separated when a species diverges into two separatespecies) or a paralog (i.e., separated by gene duplication within agenome). Typically, homologous genes encode proteins with a moderate tohigh sequence identity (e.g., at least about 30%, such as at least about50%, such as at least about 60%, such as at least about 70%, such as atleast about 80%, such as at least about 90%, such as at least about 95%,such as at least about 99%, over at least the catalytically activeportion, optionally over the full length) and/or can at least partiallysubstitute for the other protein in terms of function, when transferredfrom one species into another. Homologs of a particular gene can beidentified using publicly available and specialized biologicaldatabases, e.g., by the eggNOG, InParanoid, OrthoDB, OrthoMCL, OMA,Roundup, TreeFam, LOFT, Ortholuge, EnsemblCompara GeneTrees andHomoloGene.

A “variant” of a protein comprises one or more mutations, such as aminoacid substitutions, insertions and deletions as compared to the parentor reference enzyme. Typically, the variant has a high sequence identityto the parent or reference enzyme (e.g., at least about 70%, such as atleast about 80%, such as at least about 90%, such as at least about 91%,92%, 93%, 94%, 95%, 96%, 97% or 98%, such as at least about 99%, over atleast a catalytically active portion, optionally over the full length ofthe mature form of the reference enzyme, excluding signal peptidesequences and the like). As used herein, a variant has less than 100%sequence identity over the full-length of the mature form of the parentor reference enzyme. A catalytically active portion of Homo sapiens TPH2may, for example, correspond to residues E147 to T460 of Homo sapiensTPH2 (SEQ ID NO:3; NP_775489.2), wherein the term “corresponds to” acertain reference residue means that the residue aligns with the residuewhen using a standard dual or multiple sequence alignment program suchas, e.g., ClustalW (available at, e.g., www.genome.jp) or ClustalOmega(available at, e.g., www.ebi.ac.uk), typically using the defaultsettings.

A “fragment” of a protein comprises at least the part of the proteinwhich is responsible for its function of interest, e.g., in the case ofan enzyme, its catalytic part for the enzymatic activity of interest.Typically, a “fragment” comprises a segment corresponding to at leastabout 30%, such as at least about 50%, such as at least about 60%, suchas at least about 70%, such as at least about 80%, such as at leastabout 90%, such as at least about 95%, of the full-length protein.

A “functionally active” or “catalytically active” variant or fragmentcomprises mutations or deletions, respectively, which do notsubstantially affect the function or catalytic activity of the variantor fragment as compared to the parent or reference protein and cansubstitute at least partially for the parent or reference protein interms of the function of interest. Typically, unless used in the contextof mutations in specific TPH or GHC1 (FolE) amino acid residuesaccording to the invention, a variant or fragment has a function, asdetermined by a suitable activity assay, of 80-120%, such as 90%-110%,such as 95%-105%, of the parent or reference protein.

As used herein, “vector” refers to any genetic element capable ofserving as a vehicle of genetic transfer, expression, or replication fora exogenous nucleic acid sequence in a host cell. For example, a vectormay be an artificial chromosome or a plasmid, and may be capable ofstable integration into a host cell genome, or it may exist as anindependent genetic element (e.g., episome, plasmid). A vector may existas a single nucleic acid sequence or as two or more separate nucleicacid sequences. Vectors may be single copy vectors or multicopy vectorswhen present in a host cell. Preferred vectors for use in the presentinvention are expression vector molecules in which one or morefunctional genes can be inserted into the vector molecule, in properorientation and proximity to expression control elements resident in theexpression vector molecule so as to direct expression of one or moreproteins when the vector molecule resides in an appropriate host cell.

Construction of appropriate expression vectors and other recombinant orgenetic modification techniques for practicing the invention are wellknown in the art (see, e.g., Green and Sambrook, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory Press (Cold SpringHarbor, N.Y.) (2012), and Ausubel et al., Short Protocols in MolecularBiology, Current Protocols John Wiley and Sons (New Jersey) (2002), andreferences cited herein). Appropriate microbial cells and vectors areavailable commercially through, for example, the American Type CultureCollection (ATCC), Rockville, Md.

The term “host cell” refers to any cell into which an exogenous nucleicacid sequence can be introduced and expressed, typically via anexpression vector. The host cell may, for example, be a wild-type cellisolated from its natural environment, a mutant cell identified byscreening, a cell of a commercially available strain, or a geneticallyengineered cell or mutant cell, comprising one or more other exogenousand/or heterologous nucleic acid sequences than those of the invention.

A “recombinant” cell or host cell as used herein refers to a host cellinto which one or more exogenous nucleic acid sequences of the inventionhave been introduced, typically via transformation of a host cell with avector.

The term “substrate” or “precursor”, as used herein in relation to aspecific enzyme, refers to a molecule upon which the enzyme acts to forma product. When used in relation to an exogenous biometabolic pathway,the term “substrate” or “precursor” refers to the molecule(s) upon whichthe first enzyme of the referenced pathway acts, such as, e.g., GTP inthe pathway shown in FIG. 1. When referring to an enzyme-catalyzedreaction in a microbial cell, an “endogenous” substrate or precursor isa molecule which is native to or biosynthesized by the microbial cell,whereas an “exogenous” substrate or precursor is a molecule which isadded to the microbial cell, via a medium or the like. For example, inthe biometabolic pathway shown in FIG. 1, GTP is normally present inmicrobial host cells, and is therefore an endogenous substrate.

Unless otherwise stated, the term “sequence identity” for amino acidsequences as used herein refers to the sequence identity calculated as(n_(ref)−n_(dif))·100/n_(ref), wherein n_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinn_(ref) is the number of residues in one of the sequences. Hence, theamino acid sequence GSTDYTQNWA will have a sequence identity of 80% withthe sequence GSTGYTQAWA (n_(dif)=2 and n_(ref)=10). The sequenceidentity can be determined by conventional methods, e.g., Smith andWaterman, (1981), Adv. Appl. Math. 2:482, by the ‘search for similarity’method of Pearson & Lipman, (1988), Proc. Natl. Acad. Sci. USA 85:2444,using the CLUSTAL W algorithm of Thompson et al., (1994), Nucleic AcidsRes 22:467380, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group). The BLAST algorithm (Altschul et al., (1990),Mol. Biol. 215:403-10) for which software may be obtained through theNational Center for Biotechnology Information www.ncbi.nlm.nih.gov/) mayalso be used. When using any of the aforementioned algorithms, thedefault parameters for “Window” length, gap penalty, etc., are used.Preferably, the sequence identity between two amino acid sequences isdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofthe EMBOSS package (EMBOSS: The European Molecular Biology Open SoftwareSuite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version5.0.0 or later, e.g., as provided at World-Wide Web-addressebi.ac.uk/Tools/psalemboss_needle/. The parameters used are typically agap open penalty of 10, a gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows: (IdenticalResidues×100)/(Length of Alignment−Total Number of Gaps in Alignment).In some embodiments, sequence identity values as used herein do not takeinto account specifically identified mutations of the embodiment itrefers to, e.g., specifically identified amino acid substitutions.

Enzymes referred to herein can be classified on the basis of thehandbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYMEsite at the internet: http://www.expasy.ch/enzyme/. This is a repositoryof information relative to the nomenclature of enzymes, and is primarilybased on the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUB-MB). Itdescribes each type of characterized enzyme for which an EC (EnzymeCommission) number has been provided (Bairoch A., The ENZYME database,2000, Nucleic Acids Res 28:304-305). The IUBMB Enzyme nomenclature isbased on the substrate specificity and occasionally on their molecularmechanism.

Specific Embodiments of the Invention

As indicated in the Summary, in some aspects, the present inventionrelates to variants of TPH having improved tryptophan hydroxylaseactivity over the native parent enzyme, to variants of GCH1 (FolE)providing for improved amino acid hydroxylase activity in a microbialcell, and to variant nucleic acid sequences providing for improved,typically increased, expression of enzymes or other proteins. Thepresent invention also relates to recombinant microbial cells comprisinga heterologous monooxygenase, a heterologous PCD and, optionally, aheterologous and/or variant GCH1.

Monooxygenases

As used herein, suitable monooxygenases include, but are not limited to,phenylalanine hydroxylase (EC 1.14.16.1), phenylalanine 3-hydroxylase(EC 1.14.16.7), tyrosine 3-hydroxylase (EC 1.14.16.2), anthranilate3-monooxygenase (EC 1.14.16.3), mandelate 4-monooxygenase (EC 1.14.16.6)and alkylglycerol monooxygenase (EC 1.14.16.5). Preferably, themonooxygenase is (a) capable of catalyzing the addition of ahydroxyl-group to an aromatic ring, preferably the aromatic ring of anaromatic amino acid such as, e.g., tryptophan, tyrosine, phenylalanine,histidine, thyroxine, 5HTP and/or L-DOPA, preferably aromatic aminoacids in L-form; (b) capable of utilizing one or more of MH4, THB,tetrahydrofolate, 5-methyl-tetrahydrofolate, 5-formyl-tetrahydrofolate,10-formyl-tetrahydrofolate, 6-carboxy-tetrahydropterin, and6-hydroxymethyl-tetrahydropterin as cofactor for the referencedreaction. Particularly preferred are amino acid hydroxylases, witharomatic amino acid hydroxylases such as TPHs, THs and PheHs being mostpreferred. In a particular embodiment, the aromatic amino acidhydroxylase is not a PheH from a non-flowering plant.

Suitable TPHs, THs and PheHs include those listed in Table 1.

1. L-Tryptophan Hydroxylase (TPH)

Sources of nucleic acid sequences encoding a TPH include any specieswhere the encoded gene product is capable of catalyzing the referencedreaction, including humans, mammals such as, e.g., mouse, cow, horse,chicken and pig, as well as other animals such as, e.g., the parasiteSchistosoma mansoni. In humans and, it is believed, in other mammals,there are two distinct TPH alleles, referred to herein as TPH1 and TPH2,respectively. As used herein, a TPH is an enzyme capable of catalyzingthe hydroxylation of L-tryptophan to 5HTP.

Examples of nucleic acids encoding TPH for use in aspects andembodiments of the present invention include, but are not limited to,those encoding the TPHs listed in Table 1, as well as functionallyactive variants, homologs and fragments thereof. The amino acid sequenceof a TPH from Mesocricetus auratus (Golden hamster) is provided in NCBIReference Sequence NP 001297486.1.

Functional fragments and variants of TPH enzymes are known in the art.For example, to increase heterologous expression in E. coli and theenzyme stability, the TPH sequence can be truncated to remove portionsnot needed for its catalytic activity which preserving the catalyticcore of the enzyme. Specific examples of functional fragments of TPHinclude Met102 to Ser416 of Oryctolagus cuniculus TPH (Moran et al., JBiol Chem 1998; 273(20): 12259-66) and residues Asp45-Arg471 orGlu147-Thr460 (i.e., E147 to T460) of Homo sapiens TPH2, optionallycomprising an added N-terminal methionine residue (see SEQ ID NOS:12 and13, respectively). Other TPH sequences can be similarly truncated tocreate functionally active fragments or variants comprising thecatalytic core. For example, SEQ ID NO:11 represents a fragment of Homosapiens TPH2 comprising an added heterologous 20-amino acid polypeptideat its C-terminal. The Homo sapiens TPH2 sequence reported as NCBIaccession No. NP_775489 (SEQ ID NO:10) has a 6-amino acid insert in theN-terminal portion.

Notably, Schistosoma mansoni TPH (SEQ ID NO:9) has advantageousproperties with respect to, e.g., solubility, thus enabling no or lesstruncation of the enzyme sequence. In addition, as reported in Example3, Schistosoma mansoni TPH has advantageous catalytic activity in anoptimized recombinant cell according to the invention. Accordingly, inone embodiment, the TPH is Schistosoma mansoni TPH, or a functionallyactive fragment and/or variant thereof.

In one embodiment of any aspect of the invention, the TPH is a mammalianTPH, such as, e.g., Homo sapiens TPH2, or a fragment and/or variantthereof. In a preferred embodiment, the TPH comprises or consistsessentially of a fragment of a TPH which corresponds to (i.e., alignswith) residues E147 to T460 or L148 to T460 of Homo sapiens TPH2 (SEQ IDNO:3), typically with an N-terminal methionine (M) residue; or acatalytically active variant thereof. This particular hsTPH fragment isset forth as SEQ ID NO:13.

In one embodiment, the TPH or TPH fragment is a variant comprising oneor more mutations, such as insertions, deletions or amino acidsubstitutions, in at least one residue as compared to the reference(native parent) sequence. Preferably, a TPH variant has a mutation in aresidue corresponding to a residue selected from E147 (if present),Asp242 (N, asparagine) and Pro244 (P, proline) in Homo sapiens TPH2.

The mutation in residue corresponding to E147 is preferably an aminoacid substitution to a Lys (K; lysine), Arg (R; arginine) or His (H;histidine), most preferably an 147K mutation. Even more preferably, thenucleic acid sequence encoding the TPH has the 5′-end sequence atgaaa,encoding MK. In a specific embodiment, the nucleic acid sequence isoperatively linked to a Trc promoter, optionally comprising the nucleicacid sequence of SEQ ID NO:53. Without being limited to theory, themutation encoding for the N-terminal Met-Lys residues provides for anincreased activity and/or expression level of the TPH.

The mutation in the residue corresponding to residue N242 in Homosapiens TPH2 (SEQ ID NO:3) is preferably an amino acid substitution toIle (I; isoleucine), i.e., 242I, whereas the mutation in residue P244 ispreferably an amino acid substitution to a Cys (C, cysteine), Asp (D,aspartic acid), Leu (L, leucine) or Glu (Q, glutamine), i.e., 244C,244D, 244L or 244Q, preferably 244D or 244C, such as 244D. The variantmay also comprise a combination of two or more amino acid substitutions,such as, e.g., 242I/244D or 242I/244C, optionally in combination with147K.

In a particularly preferred embodiment, the TPH variant comprises orconsists of the amino acid sequence of SEQ ID NO:13 with E2K, N97I andP99C mutations introduced.

In another aspect, the invention provides SEQ ID NO: 13 having E2K, N97Iand P99C mutations.

Typically, the TPH variant has tryptophan hydroxylation activity and asequence identity of at least 30%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80%, such as at least90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%, such asat least 99%, over at least the catalytically active portion, optionallyover the full length, of the native TPH amino acid sequence in itsmature form, excluding signal peptide sequences and the like.Preferably, a TPH variant comprising one or more mutations according tothe invention provides for a tryptophan hydroxylation activity at leastsimilar, typically higher than of the reference TPH, typically thenative or parent TPH. For example, the TPH variant may provide for atryptophan hydroxylation activity which is at least about at least about100%, such as at least about 110%, such as at least about 130%, such asat least about 150%, such as at least about 200%, such as at least 250%of that of the native or parent TPH. Since TPH may also havephenylalanine hydroxylation activity, a TPH variant comprising one ormore mutations according to the invention may also or alternativelyprovide for a phenylalanine hydroxylation activity at least similar to,or higher than, that of the reference TPH, typically the native orparent TPH. For example, the TPH variant may provide for a phenylalaninehydroxylation activity which is at least about 90%, such as at leastabout 100%, such as at least about 110%, such as at least about 130%,such as at least about 150%, such as at least about 200%, such as atleast 250%, of that of the native or parent TPH, e.g., Homo sapiensTPH2.

Assays for measuring TPH activity in vitro are well-known in the art(see, e.g., Winge et al., Biochem J, 2008; 410:195-204 and Moran et al.,1998). Suitable assays are also provided by the present Examples, e.g.,the assays in Examples 2-4, reflecting the ability of the TPH tocatalyze the conversion of L-tryptophan to 5HTP, and Examples 6 and 7,reflecting the ability of the TPH to catalyze the conversion ofL-phenylalanine to L-tyrosine.

In the recombinant host cell, the TPH is typically sufficientlyexpressed so that an increased level of 5HTP production fromL-tryptophan can be detected as compared to the microbial host cellprior to transformation with the TPH, optionally in the presence ofadded THB cofactor and/or tryptophan substrate. Typically, in thecontext of the present invention, THB cofactor is not added.Alternatively, the expression level of the specific TPH enzyme can beevaluated by proteomic analysis, according to methods known in the art.In a preferred embodiment, the nucleic acid sequence encoding the TPH isoperably linked to a strong promoter such as the Trc promoter, providingfor high expression levels of the TPH.

2. L-Tyrosine Hydroxylase (TH)

Sources of nucleic acid sequences encoding a TH include any specieswhere the encoded gene product is capable of catalyzing the referencedreaction, including humans, mammals such as, e.g., mouse, cow, horse,chicken and pig, as well as other animals. As used herein, a TH is anenzyme capable of catalyzing the hydroxylation of L-tyrosine to L-DOPA.

Examples of nucleic acids encoding a TH for use in aspects andembodiments of the present invention include, but are not limited to,those encoding the THs listed in Table 1, as well as catalyticallyactive variants, homologs and fragments thereof. Exemplary variants andfragments of a TH include those resulting in the N-terminal sequence MK,e.g., by making an X2K amino acid substitution in a native TH having theN-terminal sequence MX (Met-Xaa), wherein X is not K (Lys), or aninsertion of a K between the N-terminal M and X amino acid residues, Inone embodiment, the TH is Rattus norwegicus TH, or a functionally activevariant, homolog or fragment thereof.

Assays for measuring TH activity in vitro are well-known in the art. Asuitable assay is also provided in the present Example 5, reflecting theability of the TH to catalyze the conversion of L-tyrosine toL-3,4-dihydroxyphenylalaine (L-DOPA).

In the recombinant host cell, the TH is typically sufficiently expressedso that an increased level of L-DOPA production from L-tyrosine can bedetected as compared to the microbial host cell prior to transformationwith the TH, optionally in the presence of added THB cofactor and/ortyrosine substrate. Typically, in the context of the present invention,THB cofactor is not added. Alternatively, the expression level of thespecific TH enzyme can be evaluated by proteomic analysis, according tomethods known in the art. In a preferred embodiment, the nucleic acidsequence encoding the TH is operably linked to a strong promoter such asthe Trc promoter, providing for high expression levels of the TH.

3. L-Phenylalanine Hydroxylase (PheH)

Sources of nucleic acid sequences encoding a PheH include any specieswhere the encoded gene product is capable of catalyzing the referencedreaction, including humans, mammals such as, e.g., mouse, cow, horse,chicken and pig, as well as other animals. As used herein, a PheH is anenzyme capable of catalyzing the hydroxylation of L-phenylalanine totyrosine.

Examples of nucleic acids encoding a PheH for use in aspects andembodiments of the present invention include, but are not limited to,those encoding the PheHs listed in Table 1, as well as functionallyactive variants, homologs and fragments thereof. Exemplary variants andfragments of a PheH include those resulting in the N-terminal sequenceMK, e.g., by making an X2K amino acid substitution in a native PheHhaving the N-terminal sequence MX, wherein X is not K, or an insertionof a K between the N-terminal M and X amino acid residues, In oneembodiment, the PheH is C. violaceum PheH, or a functionally activevariant, homolog or fragment thereof.

In a specific embodiment, the PheH is a phenylalanine 3-hydroxylase,catalyzing the conversion of phenylalanine to m-tyrosine.

Assays for measuring PheH activity in vitro are well-known in the art. Asuitable assay is also provided in the present Example 1, reflecting theability of the PheH to catalyze the conversion of L-phenylalanine toL-tyrosine, as well as Examples 6 and 7.

In the recombinant host cell, the PheH is typically sufficientlyexpressed so that an increased level of L-tyrosine (or a variantthereof, such as, e.g., L-m-tyrosine) production from L-phenylalaninecan be detected as compared to the microbial host cell prior totransformation with the PheH, optionally in the presence of added THBcofactor and/or phenylalanine substrate. Typically, in the context ofthe present invention, THB cofactor is not added. Alternatively, theexpression level of the specific PheH enzyme can be evaluated byproteomic analysis, according to methods known in the art. In apreferred embodiment, the nucleic acid sequence encoding the PheH isoperably linked to a strong promoter such as the Trc promoter, providingfor high expression levels of the PheH.

Pterin-4α-Carbolamine Dehydratase (PCD)

The PCD is typically classified as EC 4.2.1.96, and converts HTHB to DHBin the presence of water, as shown in FIG. 1. Exemplary nucleic acidsencoding PCD enzymes for use in aspects and embodiments of the presentinvention include, but are not limited to, those encoding the PCDs shownin Table 1, as well as functionally active variants, homologs andfragments thereof. In other embodiments, the exogenous nucleic acidencoding a PCD can encode a PCD which is endogenous to the microbialhost cell, e.g., in the case of host cells from Chromobacteriumviolaceum, Bacillus cereus, Lactobacillus ruminis Pseudomonas aeruginosaor Rhodobacteraceae bacterium. In some embodiments, the microbial hostcell endogenously comprises a sufficient amount of a native PCD. In suchcases, transformation of the host cell with an exogenous nucleic acidencoding a PCD is optional.

Exemplary variants and fragments of a PCD include those resulting in theN-terminal sequence MK, e.g., by making an X2K amino acid substitutionin a native PCD having the N-terminal sequence MX, wherein X is not K,or an insertion of a K between the N-terminal M and X amino acidresidues, In one embodiment, the PCD is C. violaceum PheH, or afunctionally active variant, homolog or fragment thereof. In a preferredembodiment of any aspect of the invention, the PCD comprises or consistsessentially of Chromobacterium violaceum PCD (SEQ ID NO:21) or afunctionally active variant, homolog or fragment thereof.

Assays for measuring PCD activity in vitro are well-known in the art(see, e.g., Köster et al., Biol. Chem. 1998; 379: 1427-1432). In therecombinant host cell, a sufficient expression of the PCD can typicallybe detected using an indirect assay, for example, measuringphenylalanine to tyrosine conversion of a PheH or TPH in a tyrosineauxotroph (see, e.g., Examples 1, 6 and 7). The expression level of thespecific PCD enzyme can be evaluated according to methods known in theart, e.g., by proteomic analysis. In a preferred embodiment, the nucleicacid sequence encoding the PCD is operably linked to a strong promoter,providing for high expression levels of the PCD.

GTP Cyclohydrolase I (GCH1)

Sources of nucleic acid sequences encoding a GCH1 include any specieswhere the encoded gene product is capable of catalyzing the referencedreaction. Examples of nucleic acids encoding a GCH1 for use in aspectsand embodiments of the present invention include, but are not limitedto, those encoding the GCH1 s listed in Table 1, as well as functionallyactive variants, homologs and fragments thereof. As used herein, a GCH1is an enzyme capable of catalyzing the conversion of GTP to7,8-dihydroneopterin triphosphate.

In one embodiment, the recombinant microbial cell comprises a GCH1having the same or similar catalytic activity as E. coli GCH1 (FolE; SEQID NO:16) or another GCH1 listed in Table 1, or a catalytically activefragment and/or variant thereof. Advantageously, the GCH1 is FolE or avariant of FolE. FolE is a homodecameric protein and is composed of apentamer of five dimers, and is the first enzyme in several biosyntheticpathways for production of tetrahydrofolates, tetrahydromonapterin andothers which may act as monooxygenase (e.g., TPH, TH and PheH) cofactorsin E. coli (see FIG. 1).

Surprisingly, as described in Examples 1 and 4-7, it was found thatmutations in a number of residues of E. coli GCH1 were capable ofincreasing the supply of a cofactor for TPH, PheH and TH in E. colicells. Accordingly, in one aspect of the invention, there is provided avariant of E. coli FolE (SEQ ID NO: 16) comprising one or moremutations, wherein, in an E. coli cell comprising apterin-4α-carbinolamine dehydratase (PCD) and at least one of atryptophan hydroxylase (TPH), a tyrosine hydroxylase (TH) and aphenylalanine hydroxylase (PheH), the variant provides for an increasedhydroxylation activity of at least one of the TPH, TH and PheH ascompared to native E. coli GCH1. In one embodiment, the mutation is notT198P. In another embodiment, the amino acid sequence of the variant ischaracterized by a moderate or high sequence identity to the native FolEsequence, as described below.

In one embodiment, at least one of the one or more mutations is in anamino acid residue in a segment selected from D97-E112, K121-D130,N170-H180, S193-L200 and S207-N222. For example, amino acid residues forsuch mutations include, but are not limited to, D97, M99, T101, V102,A125, K129, N170, V179, T196, T198, S199, L200, S207, H212, E213, F214,L215, H221, or a combination thereof. Preferred, non-limiting amino acidresidues for such mutations include, but are not limited to, D97, M99,N170, V179, T198, L200, S207, H212, E213, F214, L215, and H221. Mostpreferably, the amino acid residue is at least one amino acid residueselected from M99, V179, T198, L200, F214, and L215. Preferably, themutation in T198 is not T198P.

In one embodiment, exemplary amino acid substitutions in these segmentsinclude, but are not limited to, D97V, D97L, D97A, D97T, M99C, M99T,M99V, M99L, M99I, T101I, T101V, T101L, V102M, N170K, N170D, N170L,V179A, V179M, T196I, T196V, T196L, T198I, T198V, T198S, T198L, S199Y,S199F, L200P, L200C, L200S, L200A, S207R, S207K, S207M, H212R, H212K,E213K, E213R, F214A, F214G, F214S, L215P, L215Q, L215N, L215D, L215T,L215S, L215G, L215A, L215C, L215F, L215M, H221R, H221K, and combinationsthereof. Preferred, non-limiting, mutations in these amino acid residuesinclude D97V, M99I, N170K, N170D, V179A, V179M, T198I, T198S, L200P,S207R, H212R, E213K, F214S, L215Q, L215P and H221R, and combinations ofany two or more thereof. In separate and specific embodiments, thevariant comprises a mutation selected from M991, V179A, T198I, T198S,L200P, F214S, and L215P.

In another embodiment, exemplary amino acid residues for such mutationsinclude, without limitation: S3, S5, H12, A14, V28, H29, A41, G42, E46,N52, D57, M61, E62, 167, A68, 175, D97, M99, V102, T108, T117, A125,K129, I1133, Q157, N170, V179, K184, G187, T198, S199, L200, S207, H212,E213, F214, L215, H221, N222, and combinations thereof. Examples ofamino acid substitutions in these residues include, without limitation,S3L, S5C, H12R, A14V, V28L, V28A, H29Y, A41G, G42D, E46D, N52K, D57V,M61I, E62K, I67V, A68S, I75V, D97V, M99I, V102M, T108N, T171, A125D,K129N, I133F, Q157L, N170K, N170D, V179A, V179M, K184R, G187S, T198I,T198S, S199Y, L200P, S207R, H212R, E213K, F214S, L215P, L215Q, H221R,N222I, and combinations thereof.

Specific, non-limiting combinations of mutations include:

-   -   (a) I67V, T117I, A125D and H221R    -   (b) E62K, N170K and L215P    -   (c) V102M and L215P    -   (d) Q157L and H212R    -   (e) V28L, L215P and N222I    -   (f) T108N, I133F and E213K    -   (g) S5C, D57V and L215Q    -   (h) H29Y, I75V and V179M    -   (i) A14V, E46D, M61I and D97V    -   (j) V28A, G42D and E213K    -   (k) N52K, A68S and S207R    -   (l) A41G, K129N and I133F    -   (m) S3L, K184R and S199Y; and    -   (n) H12R, N170D and G187S.

Nar et al. (1995) and Rebelo et al. (2003) have reported on thestructure of FolE. The T198 residue is located in the 4^(th) β-strandthat is located in close proximity to an Ca-helix defined by Ser207 toAsn222, apparently involved in oligomerization of the FolE complex.Thus, T198 can play a role in FolE oligomerization. Specifically, themutation could affect the hydrophobic interaction with Leu215 andsubsequently the positioning of the Ser207-Asn222 helix and overallpolymerization of the FolE complex. Moreover, the alignment in FIG. 4indicates that several of these segments conserved in the Homo sapiens(SEQ ID NO:14) and S. cerevisiae (SEQ ID NO:17) homologs of FolE.

So, in another specific embodiment, there is provided a functionallyactive variant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to D97, e.g., a D97V,D97L, D97A or D97T substitution. In one embodiment, the GCH1 is E. coliGCH1 wherein, preferably, the mutation is D97V. In one embodiment, thehomolog is Homo sapiens GCH1 (SEQ ID NO:14) wherein, preferably, themutation is D127V. In one embodiment, the homolog is S. cerevisiae GCH1(SEQ ID NO:17) wherein, preferably, the mutation is D118V.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to M99, e.g., a M99C,M99T, M99V, M99L or M99I substitution. In one embodiment, the GCH1 is E.coli GCH1 wherein, preferably, the mutation is M99I. In one embodiment,the homolog is Homo sapiens GCH1 wherein, preferably, the mutation isM129I. In one embodiment, the homolog is S. cerevisiae GCH1 wherein,preferably, the mutation is M120V.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE) comprising amutation in the residue corresponding to N170, e.g., a N170K, N170D orN170L substitution. In one embodiment, the GCH1 is E. coli GCH1,wherein, preferably, the mutation is N170K or N170D. In one embodiment,the homolog is Homo sapiens GCH1 wherein, preferably, the mutation isA200K or A200D. In one embodiment, the homolog is S. cerevisiae GCH1,and the mutation is L191K or L191D.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to V179, e.g., aV179A or V179M substitution. In one embodiment, the GCH1 is E. coli GCH1wherein, preferably, the mutation is V179A. In one embodiment, thehomolog is Homo sapiens GCH1 wherein, preferably, the mutation is T209A.In one embodiment, the homolog is S. cerevisiae GCH1, and the mutationis S200A.

In a specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to T198, e.g., aT198S, T198I, T198V or T198L substitution. In a specific embodiment, theT198 substitution is not T198P. In one embodiment, the GCH1 is E. coliGCH1 wherein, preferably, the mutation is T198S or T198I. In oneembodiment, the homolog is Homo sapiens GCH1 wherein, preferably, themutation is S228I. In one embodiment, the homolog is S. cerevisiae GCH1wherein, preferably, the mutation is S219I.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to L200, e.g., aL200P, L200C, L200S or L200A substitution. In one embodiment, the GCH1is E. coli GCH1 wherein, preferably, the mutation is L200P. In oneembodiment, the homolog is Homo sapiens GCH1 wherein, preferably, themutation is M230P. In one embodiment, the homolog is S. cerevisiae GCH1wherein, preferably, the mutation is M221P.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO: 16)comprising a mutation in the residue corresponding to S207, e.g., aS207R, S207K, or S207M substitution. In one embodiment, the GCH1 is E.coli GCH1 wherein, preferably, the mutation is S207R. In one embodiment,the homolog is Homo sapiens GCH1 wherein, preferably, the mutation isD237R. In one embodiment, the homolog is S. cerevisiae GCH1, and themutation is H228R.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to H212, e.g., aH212R or H212K substitution. In one embodiment, the GCH1 is E. coli GCH1wherein, preferably, the mutation is H212R. In one embodiment, thehomolog is Homo sapiens GCH1 wherein, preferably, the mutation is E242R.In one embodiment, the homolog is S. cerevisiae GCH1, and the mutationis E232R.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to E213, e.g., aE213K or E213R substitution. In one embodiment, the GCH1 is E. coli GCH1wherein, preferably, the mutation is E213K. In one embodiment, thehomolog is Homo sapiens GCH1 wherein, preferably, the mutation is E243K.In one embodiment, the homolog is S. cerevisiae GCH1 wherein,preferably, the mutation is E233K.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to F214, e.g., aF214A, F214G or F214S substitution. In one embodiment, the GCH1 is E.coli GCH1 wherein, preferably, the mutation is F214S. In one embodiment,the homolog is Homo sapiens GCH1 wherein, preferably, the mutation isF244S. In one embodiment, the homolog is S. cerevisiae GCH1 wherein,preferably, the mutation is F234S.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to L215, e.g., aL215P, L215Q, L215N, L215D, L215T, L215S, L215G, L215A, L215C, L215F orL215M substitution. In one embodiment, the GCH1 is E. coli GCH1 wherein,preferably, the mutation is L215P or L215Q. In one embodiment, thehomolog is Homo sapiens GCH1 wherein, preferably, the mutation is L245Por L245Q. In one embodiment, the homolog is S. cerevisiae GCH1 wherein,preferably, the mutation is L235P or L235Q.

In another specific embodiment, there is provided a functionally activevariant, homolog or fragment of E. coli GCH1 (FolE; SEQ ID NO:16)comprising a mutation in the residue corresponding to H221, e.g., aH221R or H221K substitution. Preferably, the mutation in FolE is H221R.In one embodiment, the GCH1 is S. cerevisiae GCH1, and the mutation isH241K.

In a specific embodiment, a GCH1 variant of any preceding embodimentcomprises the native, mature GCH1 sequence except for the listedmutation(s).

In one embodiment, any E. coli GCH1 (FolE) variant or fragment retainthe native residues corresponding to C111, C182, H113 and H114 of theenzyme since, as reported by Rebelo (2003), replacement of theseresidues made the enzyme catalytically inactive.

In another aspect, the invention provides FolE (SEQ ID NO: 16) having amutation selected from D97V, M99I, N170K, N170D, V179A, V179M, T198I,T198S, L200P, S207R, H212R, E213K, F214S, L215Q, L215P and H221R, or acombination of any two or more thereof.

In another aspect, the invention provides Homo sapiens GCH1 (SEQ IDNO:14) having a mutation selected from D127V, M129I, A200K, A200D,T209A, S228I, M230P, D237R, E242R, E243K, F244S, L245P and L245Q, or acombination selected from any two or more thereof.

In another aspect, the invention provides S. cerevisiae GCH1 (SEQ IDNO:17) having a mutation selected from D118V, M120V, L191K, L191D,S200A, S219I, M221P, H228R, E232R, E233K, F234S, L235P, L235Q and H241K,or a combination selected from any two or more thereof.

Typically, the functionally active GCH1 (FolE; SEQ ID NO: 16) varianthas a sequence identity of at least 30%, such as at least 50%, such asat least 60%, such as at least 70%, such as at least 80%, such as atleast 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%,such as at least 99%, over at least the catalytically active portion,optionally over the full length, of the reference or native GCH1 aminoacid sequence in its mature form, excluding signal peptide sequences andthe like. In some embodiments, the sequence identity value for a GCH1variant does not take into account any specifically identified mutationin the embodiment in question. In one embodiment, the reference ornative GHC1 sequence is SEQ ID NO:16 (FolE). In one embodiment, thereference or native sequence is SEQ ID NO:14 (Homo sapiens GCH1). In oneembodiment, the reference or native sequence is SEQ ID NO:17 (S.cerevisiae GCH1).

The GCH1 variants of the invention may provide for one, two or all of atryptophan, tyrosine and phenylalanine hydroxylation activity which isat least similar to, typically higher than that of the reference GCH1,e.g., the native or parent GCH1. This can be investigated in a microbialcell comprising the GCH1 variant, the hydroxylase and a PCD.

In one embodiment, a GCH1 variant comprising mutations in one or morespecific amino acid residues or one or more specific amino acidsubstitutions according to the invention provides for a tryptophanhydroxylation activity at least similar to, typically higher than thatof the reference GCH1, typically the native or parent GCH1, in amicrobial cell comprising a TPH. For example, such a GCH1 variant may,in an E. coli cell comprising a TPH and a PCD provide for a tryptophanhydroxylase activity, converting L-tryptophan to 5HTP, which is at leastabout 100%, such as at least about 110%, such as at least about 130%,such as at least about 150%, such as at least about 200%, such as atleast about 250%, such as at least about 300% of that of the native orparent GCH1. Suitable assays are provided in the Examples, e.g., Example4. For example, the TPH may be Homo sapiens TPH2 (SEQ ID NO:3) or afunctionally active variant, homolog or fragment thereof, such as a TPHvariant comprising or consisting of the amino acid sequence of SEQ IDNO:13 with E2K, N97I and P99C mutations introduced; the PCD may, forexample, be C. violaceum PCD (SEQ ID NO:21) or a functionally activevariant, homolog or fragment thereof; and the assay conducted in thepresence of about 100 mg/L L-tryptophan.

In one embodiment, a GCH1 variant comprising mutations in one or morespecific amino acid residues or one or more specific amino acidsubstitutions according to the invention provides for a tyrosinehydroxylation activity at least similar to, typically higher than thatof the reference GCH1, typically the native or parent GCH1, in amicrobial cell comprising a TH. For example, such a GCH1 variant may, inan E. coli cell comprising a TH and a PCD provide for a tyrosinehydroxylase activity, converting L-tyrosine to L-DOPA, which is at leastabout 100%, such as at least about 110%, such as at least about 130%,such as at least about 150%, such as at least about 200%, such as atleast about 250%, such as at least about 300% of that of the native orparent GCH1. Suitable assays are provided in the Examples, e.g., Example5. For example, the TPH may be Rattus norvegicus TH (SEQ IDS NO:52) or afunctionally active variant, homolog or fragment thereof; the PCD may,for example, be C. violaceum PCD (SEQ ID NO:21) or a functionally activevariant, homolog or fragment thereof, and the assay conducted in thepresence of about 100 mg/L L-tyrosine.

In one embodiment, a GCH1 variant comprising mutations in one or morespecific amino acid residues or one or more specific amino acidsubstitutions according to the invention provides for a phenylalaninehydroxylation activity at least similar to, typically higher than thatof the reference GCH1, typically the native or parent GCH1, in amicrobial cell comprising a PheH. For example, such a GCH1 variant may,in an E. coli cell comprising a PheH (which may also be a TPH havingphenylalanine hydroxylation activity), and a PCD provide for aphenylalanine hydroxylase activity, converting L-phenylalanine totyrosine, which is at least about 100%, such as at least about 110%,such as at least about 130%, such as at least about 150%, such as atleast about 200%, such as at least about 250%, such as at least about300% of that of the native or parent GCH1. Suitable assays are providedin the Examples, e.g., Examples 1, 6 and 7. For example, the PheH may beC. violaceum PheH (SEQ ID NO:58) or a functionally active variant,homolog or fragment thereof; the PCD may, for example, be C. violaceumPCD (SEQ ID NO:21) or a functionally active variant, homolog or fragmentthereof, and the assay conducted in the presence of about 100 mg/LL-phenylalanine. Alternatively, the PheH may be a TPH also having PheHactivity, such as Homo sapiens TPH2 (SEQ ID NO:3) or a functionallyactive variant, homolog or fragment thereof, such as a TPH variantcomprising or consisting of the amino acid sequence of SEQ ID NO:13 withE2K, N97I and P99C mutations introduced.

In another aspect, the invention provides a method of selecting and,optionally, producing, a variant GCH1, the method comprising the stepsof

-   -   (a) preparing a population of variants of a parent GCH1;    -   (b) preparing a population of microbial cells, each expressing a        variant GCH1, a PCD, and at least one hydroxylase selected from        a TPH, a TH, and a PheH;    -   (c) analyzing the population of microbial cells for        hydroxylation of a substrate for the hydroxylase as compared to        a control, optionally wherein the control is a microbial cell        expressing parent GCH1, the PCD and the at least one        hydroxylase;    -   (d) selecting at least one variant GCH1 which provides increased        hydroxylation as compared to the control; and    -   (e) optionally, producing the variant GCH1 by recombinant        expression in a host cell.

Optionally, the method may comprise features of other embodiments hereinwhich describe activity testing of variant E. coli GCH1. Suitablesources of parent GCH1 include, for example, those listed in Table 1,for example Homo sapiens or S. cerevisiae GCH1. The invention alsoprovides a variant GCH1 obtained or obtainable from the method.

A mutated GCH1 according to any aspect or embodiment herein can, forexample, be expressed from an exogenously introduced nucleic acidsequence. In one embodiment, the mutated GCH1 is expressed from anucleic acid sequence integrated into the chromosome. Alternatively, thenative gene can be mutated in situ, i.e., in the chromosome of themicrobial cell, after which the mutated GCH1 is expressed from themutated gene. In some embodiments, the microbial cell expresses a lowerlevel of native GCH1 than the (native) parent cell, e.g., bydownregulating, deleting or otherwise inactivating the native GCH1 gene(e.g., folE).

In an alternative embodiment, the recombinant microbial cell comprisesan overexpressed GCH1 selected from the GCH1 enzymes listed in Table 1,optionally with a mutation corresponding to D97V, M991, N170K, N170D,V179A, V179M, T198I, T198S, L200P, S207R, H212R, E213K, F214S, L215Q,L215P or H221R, or a combination thereof.

In a preferred embodiment, the nucleic acid sequence encoding the GCH1is operably linked to a strong promoter, providing for high expressionlevels of the GCH1. Even more preferably, the nucleic acid sequenceencoding the GCH1 has the 5′-end sequence atgaaa, encoding MK. In aspecific embodiment, the nucleic acid sequence is operatively linked toa Trc promoter, optionally comprising the nucleic acid sequence of SEQID NO:53. Without being limited to theory, the mutation encoding for theN-terminal Met-Lys residues provides for an increased activity and/orexpression level of the GCH1.

In the recombinant host cell, the GCH1 is typically sufficientlyexpressed so that an increased level of 5HTP production fromL-tryptophan can be detected in the assay according to Example 2,Example 3 or Example 4, i.e., in an E. coli host cell comprising a Homosapiens TPH2 fragment or variant as described herein or a Schistosomamansoni TPH, and a C. violaceum PCD, as compared to the microbial hostcell prior to transformation with a heterologous GCH1 or prior to theintroduction of a GCH1, optionally in the presence of added tryptophansubstrate. Alternatively, the expression level of the specific GCH1enzyme can be evaluated by proteomic analysis, according to methodsknown in the art.

Downstream Enzymes

In some embodiments, particularly when the monooxygenase is a TH, therecombinant microbial cell can further comprise nucleic acid sequences,e.g., heterologous, encoding a dopa decarboxylase, a tyramine oxidaseand an alcohol dehydrogenase for production of hydroxytyrosol; e.g., asdescribed by Satoh et al. (2012).

In some embodiments, particularly when the monooxygenase is a TPH andserotonin, N-acetyl-serotonin or melatonin is a desired end-product, therecombinant microbial cell can further comprise heterologous nucleicacid sequences encoding a 5HTP decarboxylase (ADDC), a serotoninacetyltransferase (AANAT) and/or an acetylserotonin O-methyltransferase(ASMT):

1. 5HTP Decarboxylase:

The last step in the serotonin biosynthesis via a 5HTP intermediate, theconversion of 5HTP to serotonin, is in animal cells catalyzed by a 5HTPdecarboxylase, which is an aromatic L-amino acid decarboxylase (AADC)typically classified as EC 4.1.1.28. Suitable 5HTP decarboxylasesinclude any tryptophan decarboxylase (TDC) capable of catalyzing thereferenced reaction. TDC participates in the plant serotoninbiosynthesis pathway, where tryptophan decarboxylase (TDC) catalyzes theconversion of tryptophan to tryptamine, which is then converted intoserotonin in a reaction catalyzed by tryptamine 5-hydroxylase (T5H). TDClikewise belongs to the aromatic amino acid decarboxylases categorizedin EC 4.1.1.28, and can be able to convert 5HTP to serotonin and carbondioxide (see, e.g., Park et al., Biosci. Biotechnol. Biocem. 2008;72(9):2456-2458.2008, and Gibson et al., J. Exp. Bot. 1972;23(3):775-786), and thus function as a 5HTP decarboxylase. Exemplarynucleic acids encoding ADDC enzymes for use in aspects and embodimentsof the present invention include, but are not limited to, those encodingthe 5HTP decarboxylases listed in Table 1, as well as functionallyactive variants, homologs and fragments thereof. In some embodiments,particularly where it is desired to also promote serotonin formationfrom a tryptamine substrate in the same recombinant cell, an enzymecapable of catalyzing both the conversion of tryptophan to tryptamineand the conversion of 5HTP to serotonin can be used. For example, riceTDC and tomato TDC can function also as a 5HTP decarboxylase, anactivity which can be promoted by the presence of pyridoxal phosphate(e.g., at a concentration of about 0.1 mM) (Park et al., 2008).

Suitable assays for testing serotonin production by a 5HTP decarboxylasein a recombinant microbial host cell are provided in, or can be adaptedfrom, e.g., Park et al. (2008) and Park et al., Appl MicrobiolBiotechnol 2011; 89(5):1387-1394. For example, these assays can beadapted to test serotonin production by a 5HTP decarboxylase (e.g., aTDC), either from 5HTP or, in case the microbial cell comprises anL-tryptophan hydroxylase, from L-tryptophan (or simply a carbon source).In one exemplary embodiment, the recombinant microbial cell produces atleast 5%, such as at least 10%, such as at least 20%, such as at least50%, such as at least 100% or more serotonin than the recombinant cellwithout transformation with 5HTP decarboxylase enzyme, i.e., abackground value.

2. Serotonin Acetyltransferase (AANAT):

In one aspect, the recombinant microbial cell further comprises anexogenous nucleic acid sequence encoding a serotonin acetyltransferase,also known as serotonin —N-acetyltransferase, arylalkylamineN-acetyltransferase and AANAT, and typically classified as EC 2.3.1.87.AANAT catalyzes the conversion of acetyl-CoA and serotonin to CoA andN-Acetyl-serotonin. Exemplary nucleic acids encoding AANAT enzymes foruse in aspects and embodiments of the present invention include, but arenot limited to, those encoding the AANATs shown in Table 1, as well asfunctionally active variants, homologs or fragments thereof. Suitableassays for testing N-acetylserotonin production by an AANAT in arecombinant microbial host cell are described in, e.g., Thomas et al.,Analytical Biochemistry 1990; 184:228-34.

3. Acetylserotonin O-Methyltransferase (ASMT):

In one aspect, the recombinant cell further comprises an exogenousnucleic acid encoding an acetylserotonin O-methyltransferase or ASMT,typically classified as EC 2.1.1.4. ASMT catalyzes the last reaction inthe production of melatonin from L-tryptophan, the conversion ofN-acetyl-serotonin and S-adenosyl-L-methionine (SAM) to Melatonin andS-adenosyl-L-homocysteine (SAH). SAH can then be recycled back to SAMvia the S-adenosyl-L-methionine cycle in microbial cells where theS-adenosyl-L-methionine cycle is native (or exogenously added) andconstitutively expressed, such as, e.g., in E. coli. Exemplary nucleicacids encoding ASMT enzymes for use in aspects and embodiments of thepresent invention include, but are not limited to, those encoding ASMTsshown in Table 1, as well as functionally active variants, homologs orfragments thereof. Suitable assays for testing melatonin production byan ASMT in a recombinant microbial host cell have been described in,e.g., Kang et al. J. Pineal Res. 2011:50; 304-309, which is herebyincorporated by reference in its entirety.

Nucleic Acids and Vectors

In other aspects, there is provided nucleic acid sequences and vectorscomprising such nucleic acid sequences. For example, a nucleic acidsequence encoding an enzyme or other protein activity listed in Table 1or a fragment or variant thereof as described in any aspect orembodiment herein may encode an amino acid sequence that is homologous(i.e., native) or heterologous to the recombinant host cell in question,e.g., a variant or fragment of an endogenous amino acid sequence.

Also provided are one or more vectors comprising nucleic acid sequencesaccording to the above aspects and embodiments, e.g., encoding one ormore of, such as one, two, three, four, five, six or all of, amonooxygenase (e.g., TPH/TH/PheH), PCD, GCH1, 5HTP decarboxylase, AANATand ASMT, optionally wherein the GCH1 is mutant FolE as described hereinand/or the TPH is Schistosoma mansoni or variant Homo sapiens TPH2 asdescribed herein.

In a particular aspect, the invention provides a nucleic acid sequencecomprising a Trc promoter and a DNA coding sequence encoding an enzymeor other protein, wherein the 3′-end of the Trc promoter is operably,preferably directly, linked to the 5′-end of the DNA coding sequence,and wherein the 5′-end sequence of the DNA coding sequence is atgaaa.

In one embodiment, there is provided a vector comprising such a nucleicacid sequence.

In one embodiment, there is provided a method for expressing an enzymeor other protein, comprising transforming an E. coli host cell with thevector of the preceding embodiment, expressing the enzyme or otherprotein; and, optionally, harvesting the enzyme or other protein.

Preferably, in any of the preceding embodiments, the Trc promoter isdirectly linked to the DNA coding sequence. Most preferably, the nucleicacid sequence comprises SEQ ID NO:53, i.e., the DNA sequence of a Trcpromoter directly linked to atgaaa. The N-terminal amino acids of theenzyme or other protein encoded by the DNA coding sequence are MK(Met-Lys). Accordingly, the enzyme or other protein encoded by the DNAcoding sequence may be a native enzyme or protein whose N-terminal aminoacid sequence is MK. Alternatively, the enzyme or other protein may be avariant or fragment of a native enzyme or protein, wherein theN-terminal amino acid sequence is modified to MK, e.g., introducing anX2K amino acid substitution, where “X” represents the amino acidadjacent to the N-terminal methionine residue. In one embodiment, theenzyme encoded is one that is listed in Table 1 or a functionally activefragment or variant thereof, wherein the N-terminal amino acids are MK.

In one embodiment, there is provided a method of producing a recombinantvariant of a parent enzyme or other parent protein in an E. coli hostcell, wherein the N-terminal amino acid sequence of the parent enzyme orother parent protein is MX, wherein X is not lysine, the methodcomprising the steps of:

(a) preparing a vector comprising a Trc promoter operably, preferablydirectly, linked to a DNA coding sequence encoding a variant of theparent enzyme or other variant protein which has an X2K amino acidsubstitution or an insertion of a K between the N-terminal M and X,wherein the 5′-end sequence of the DNA coding sequence is atgaaa;

(b) transforming an E. coli host cell with the vector of (a);

(c) expressing the recombinant variant of the parent enzyme or otherparent protein in the E. coli host cell; and,

(d) optionally, harvesting the recombinant variant of the parent enzymeor other parent protein.

In one embodiment, the only different between the parent and variantenzyme or protein is the X2K amino acid substitution or the insertion ofa K between the N-terminal M and X. This method may provide an increasedexpression level of the variant enzyme/protein as compared to the parentenzyme/protein in E. coli cells under conditions that are otherwise thesame or similar.

The specific design of the vector according to any aspect or embodimentdepends on, e.g., whether host cell already endogenously producessufficient amounts of one or more of the enzymes. For example, in an E.coli host cell, it may not be necessary to introduce the nucleic acidsequence encoding a mutated FolE sequence exogenously, in case thenative gene can be mutated in situ to introduce the mutation (seeExample 1). Additionally, for transformation of a particular host cell,two or more vectors with different combinations of the enzymes used inthe present invention can be applied. The vector can be a plasmid, phagevector, viral vector, episome, an artificial chromosome or otherpolynucleotide construct, and may, for example, include one or moreselectable marker genes and appropriate expression control sequences.

Generally, regulatory control sequences are operably linked to theencoding nucleic acid sequences, and include constitutive, regulatoryand inducible promoters, transcription enhancers, transcriptionterminators, and the like which are well known in the art. The encodingnucleic acid sequences can be operationally linked to one commonexpression control sequence or linked to different expression controlsequences, such as one inducible promoter and one constitutive promoter.

The procedures used to ligate the various regulatory control and markerelements with the encoding nucleic acid sequences to construct thevectors of the present invention are well known to one skilled in theart (see, e.g., Sambrook et al., 2012, supra). In addition, methods haverecently been developed for assembling of multiple overlapping DNAmolecules (Gibson et al., 2008) (Gibson et al., 2009) (Li & Elledge,2007), allowing, e.g., for the assembly multiple overlapping DNAfragments by the concerted action of an exonuclease, a DNA polymeraseand a DNA ligase.

The promoter sequence is typically one that is recognized by theintended host cell. For an E. coli host cell, suitable promotersinclude, but are not limited to, the lac promoter, the T7 promoter,pBAD, the tet promoter, the Lac promoter, the Trc promoter, the Trppromoter, the recA promoter, the λ (lamda) promoter, and the PLpromoter. Preferred promoters include the Trc promoter. For Streptomyceshost cells, suitable promoters include that of Streptomyces coelicoloragarase (dagA). For a Bacillus host cell, suitable promoters include thesacB, amyL, amyM, amyQ, penP, xylA and xylB. Other promoters forbacterial cells include prokaryotic beta-lactamase (Villa-Kamaroff etal., 1978, Proceedings of the National Academy of Sciences USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proceedings ofthe National Academy of Sciences USA 80: 21-25). For an S. cerevisiaehost cell, useful promoters include the TEF1, HXT7, TDH3, ENO-1, GAL1,ADH1, ADH2, GAP, TPI, CUP1, PHO5 and PGK, such as PGK1 promoters. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8: 423-488. Still other useful promoters for various hostcells are described in “Useful proteins from recombinant bacteria” inScientific American, 1980, 242: 74-94; and in Sambrook et al., 2012,supra.

In one embodiment, one or more or all of the exogenous nucleic acids iseach under the control of a strong promoter, e.g., each separatelyselected from T7, lac, trac and PL in an E. coli host cell, and eachseparately selected from PGK1, TEF1, HXT7 and TDH3 in an S. cerevisiaehost cell.

A transcription terminator sequence is a sequence recognized by a hostcell to terminate transcription, and is typically operably linked to the3′ terminus of an encoding nucleic acid sequence. Suitable terminatorsequences for E. coli host cells include the T7 terminator region.Suitable terminator sequences for yeast host cells such as S. cerevisiaeinclude CYC1, PGK, GAL, ADH, AOX1 and GAPDH. Other useful terminatorsfor yeast host cells are described by Romanos et al., 1992, supra.

A leader sequence is a non-translated region of an mRNA which isimportant for translation by the host cell. The leader sequence istypically operably linked to the 5′ terminus of a coding nucleic acidsequence. Suitable leaders for yeast host cells include S. cerevisiaeENO-1, PGK, alpha-factor, ADH2/GAP, TEF, and Kozak sequence.

A polyadenylation sequence is a sequence operably linked to the 3′terminus of a coding nucleic acid sequence which, when transcribed, isrecognized by the host cell as a signal to add polyadenosine residues totranscribed mRNA. Useful polyadenylation sequences for yeast host cellsare described by Guo and Sherman, 1995, Mol Cell Biol 15: 5983-5990.

A signal peptide sequence encodes an amino acid sequence linked to theamino terminus of an encoded amino acid sequence, and directs theencoded amino acid sequence into the cell's secretory pathway. In somecases, the 5′ end of the coding nucleic acid sequence may inherentlycontain a signal peptide coding region naturally linked in translationreading frame, while a foreign signal peptide coding region may berequired in other cases. Useful signal peptides for yeast host cells canbe obtained from the genes for S. cerevisiae alpha-factor and invertase.Other useful signal peptide coding regions are described by Romanos etal., 1992, supra. An exemplary signal peptide for an E. coli host cellcan be obtained from alkaline phosphatase. For a Bacillus host cell,suitable signal peptide sequences can be obtained from alpha-amylase andsubtilisin. Further signal peptides are described by Simonen and Palva,1993, Microbiological Reviews 57: 109-137.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tec, and tip operator systems. For example, one or more promotersequences can be under the control of an IPTG inducer, initiatingexpression of the gene once IPTG is added. In yeast, the ADH2 system orGAL1 system may be used. Other examples of regulatory sequences arethose which allow for gene amplification. In eukaryotic systems, theseinclude the dihydrofolate reductase gene which is amplified in thepresence of methotrexate, and the metallothionein genes which areamplified with heavy metals. In these cases, the respective encodingnucleic acid sequence would be operably linked with the regulatorysequence.

The choice of the vector will typically depend on the compatibility ofthe vector with the host cell into which the vector is to be introduced.The vectors may be linear or closed circular plasmids. The vector mayalso be an autonomously replicating vector, i.e., a vector which existsas an extrachromosomal entity, the replication of which is independentof chromosomal replication, e.g., a plasmid, an extrachromosomalelement, a minichromosome, or an artificial chromosome. The vector maycontain any means for assuring self-replication. Alternatively, thevector may be one which, when introduced into the host cell, isintegrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Theselectable marker genes can, for example, provide resistance toantibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media, and/or provide for controlof chromosomal integration. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

The vectors of the present invention may also contain one or moreelements that permit integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome. For integration into the host cell genome, the vector may relyon an encoding nucleic acid sequence or other element of the vector forintegration into the genome by homologous or nonhomologousrecombination. Alternatively, the vector may contain additionalnucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. The integrationalelements may, for example, non-encoding or encoding nucleotidesequences. The vector may be integrated into the genome of the host cellby non-homologous recombination. For example, for integration into an E.coli chromosome, the vector may contain elements directing integrationof the nucleic acid sequences using the Tn7 site-specific integrationmethod according to McKenzie G and Nancy LC (2006).

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB1 10, pE194, pTA1060, andpAMβi permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6.

More than one copy of the nucleic acid sequence encoding the enzyme orprotein of interest may be inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of theencoding nucleic acid sequence can be obtained by integrating at leastone additional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the sequence, can beselected for by cultivating the cells in the presence of the appropriateselectable agent.

Recombinant Cells

The present invention relates to recombinant microbial cells comprisinga monooxygenase and a PCD. Optionally, one or both enzymes areheterologous to the microbial cell. It has now been discovered that theproduction of 5HTP, L-DOPA, tyrosine (e.g., m-tyrosine) and othercompounds that are the products of monooxygenase-catalyzed reactions insuch recombinant microbial cells can be optimized by modifications suchas omitting certain enzymes and/or introducing certain mutations incertain endogenous or exogenous nucleic acid sequences. Thesemodifications include mutations in the monooxygenase (e.g., a TPH) or ina GTP cyclohydrolase 1 (GCH1) as described in previous embodiments,and/or mutations in YnbB, mutations, downregulation or deletion of tnaA,and combinations of any two or more such modifications.

The aforementioned mutations can advantageously be combined in therecombinant host cell, particularly when the host cell is of theEscherichia genus, such as an E. coli cell. Any monooxygenase, e.g., anamino acid hydroxylase, can be used. In some embodiments, however, themonooxygenase is a TPH, a TH or a PheH. So, in one embodiment, therecombinant microbial cell comprises a PCD and one or more of (a) anucleic acid sequence encoding E. coli GTP cyclohydrolase 1 (folE) or afunctionally active variant, homolog or fragment thereof, comprising amutation as described herein, e.g., in T198, F214, V179, M99 and/orL200; and (b) a nucleic acid sequence encoding a mammalian (such as aHomo sapiens) TPH or Schistosoma mansoni TPH or a functionally activevariant, homolog or fragment thereof. In preferred embodiments, themutation in FolE is selected from T198I, T198S, F214S, V179A, M99I andL200P and/or the TPH comprises or consists of the residues correspondingto residues E147 to T460 of Homo sapiens TPH2, an N-terminal methionineresidue, and a mutation corresponding to E147K, N242I or P244D/P244C ora combination thereof such as N242I and P244D.

In one embodiment, the recombinant microbial cell, particularly when thecell is an E. coli cell, also or alternatively comprises an YnbB or afunctionally active variant, homolog or fragment thereof, comprising amutation in the residue corresponding to V197. Preferably, the mutationin V197 is V197A. The E. coli YnbB sequence is provided in SEQ ID NO:52.

In one embodiment, particularly when the monooxygenase is a TPH, therecombinant microbial cell of the invention comprises a deletion ordownregulation of an endogenous gene encoding an enzyme providing fordegradation of tryptophan, e.g., a tryptophanase (EC 4.1.99.1).

In one embodiment, the microbial cell is genetically modified, typicallymutated, to downregulate or delete tryptophanase activity. Tryptophanaseor tryptophan indole-lyase (EC 4.1.99.1), encoded by the tnaA gene in E.coli, catalyzes the hydrolytic cleavage of L-tryptophan to indole,pyruvate and NH4⁺. Active tryptophanase consists of four identicalsubunits, and enables utilization of L-tryptophan as sole source ofnitrogen or carbon for growth together with a tryptophan transporterencoded by tnaC gene. Tryptophanase is a major contributor towards thecellular L-cysteine desulfhydrase (CD) activity. In vitro, tryptophanasealso catalyzes α, β elimination, β replacement, and α hydrogen exchangereactions with a variety of L-amino acids. Tryptophan degradationmechanisms are known to also exist in other microorganisms. Forinstance, in S. cerevisiae, there are two different pathways for thedegradation of tryptophan (The Erlich pathway and the kynureninepathway, respectively), involving in their first step the aromatic aminoacid aminotransferase ARO8, ARO9, ARO10, and/or BNA2 genes.

Accordingly, reducing tryptophan degradation can be achieved by, e.g., asite-directed mutation in or deletion of a gene encoding atryptophanase, such as the tnaA gene (in E. coli or other organisms suchas Enterobacter aerogenes) (Uniprot P0A853), or the kynA gene (inBacillus species) (Uniprot Q736W5), or one or more of the ARO8 (UniprotP53090), ARO9 (Uniprot P38840), ARO10 (Uniprot Q06408) and BNA2 (UniprotP47125) genes (in S. cerevisiae). In one embodiment, the ARO9 gene isdownregulated, optionally deleted. Alternatively, tryptophanase activitycan be reduced reducing the expression of the gene by introducing amutation in, e.g., a native promoter element, or by adding an inhibitorof the tryptophanase.

In addition, as discovered by the present inventor, for the purpose ofachieving 5HTP production in a microbial cell, it is not necessary thatthe cell comprises exogenous nucleic acid sequences providing for theexpression of dihydropteridine reductase (DHPR) and/or dihydromonapterinreductase (DHMR). It is further not necessary for the cell to compriseexogenous nucleic sequences providing for the expression of6-pyruvoyl-tetrahydropterin synthase (PTPS or PTS) and/or sepiapterinreductase (SPR). Without being limited to theory, this provides forincreased metabolic resources being available for the expression of TPHand PCD, the production of 5HTP production, or both, thus improving theyield of 5HTP.

DHPR is typically classified as EC 1.5.1.34, and can convert quinonoiddihydrobiopterin (q-H₂—BPt or DHB) to tetrahydrobiopterin (H₄—BPt orTHB) in the presence of cofactor NADH. DHMR is typically classified asEC 1.5.1.50 and can convert 7,8-dihydromonapterin to5,6,7,8-tetrahydromonapterin. The endogenous E. coli DHMR gene isdesignated folM. So, in one embodiment, the recombinant microbial cellof any one of the preceding aspects or embodiments does not comprise oneor both of a heterologous or overexpressed endogenous DHPR and DHMR. Ina specific embodiment, the microbial cell does not comprise an exogenousnucleic acid sequence, such as a heterologous or overexpressedendogenous nucleic acid sequence, encoding a DHPR or a DHMR. In oneembodiment, the microbial cell does not comprise a nucleic acid encodinga DHPR or a DHMR. In an additional or alternative embodiment, themicrobial cell does not comprise a nucleic acid sequence encoding E.coli oxygen-insensitive nitroreductase (NfsB).

PTS is typically classified as EC 4.2.3.12, and can convert7,8-dihydroneopterin 3′-triphosphate ((H₂—N₃P or DHP) to6-pyruvoyltetrahydropterin (H₄-PPt or 6PTH). SPR is typically classifiedas EC 1.1.1.153, and can convert 6PTH to THB in the presence of itscofactor NADPH.

So, in one embodiment, the recombinant microbial cell of any precedingaspect or embodiment does not comprise one or both of a heterologous oroverexpressed endogenous PTPS and SPR. In a specific embodiment, themicrobial cell does not comprise an exogenous nucleic acid sequence,such as a heterologous or overexpressed endogenous nucleic acidsequence, encoding a PTS or an SPR. In another specific embodiment, themicrobial cell does not comprise a nucleic acid sequence encoding a PTSor an SPR.

In yet another embodiment, the recombinant microbial cell of anypreceding aspect or embodiment does not comprise any heterologous oroverexpressed endogenous DHPR, DHMR, PTPS or SPR. In a specificembodiment, the microbial cell does not comprise exogenous nucleic acidsequences, such as heterologous or overexpressed endogenous nucleic acidsequences, encoding a DHPR, a DHMR, PTS or SPR. In another specificembodiment, the microbial cell does not comprise nucleic acid sequencesencoding a DHPR, a DHMR, a PTS or an SPR.

TABLE 1 Examples of enzymes and amino acid sequences SEQ ID # (GenBankor Name (EC #) Species UniProtKB #) L-tryptophan Oryctolagus cuniculusTPH1 (“ocTPH”)  1 (P17290-1, v2) hydroxylase (EC Homo sapiens TPH1(“hsTPH1”)  2 (NP_004170.1) 1.14.16.4) (TPH) Homo sapiens TPH2(“hsTPH2”)  3 (NP_775489.2, Q8IWU9) Bos taurus (“bsTPH”)  4 Sus scrofa(“scTPH”)  5 Gallus gallus (“ggTPH”)  6 (NP_990287.1) Mus musculus(“mmTPH”)  7 (NP_033440.1) Equus caballus (“ecTPH”)  8 (NP_001075252.1)Schistosoma mansoni (“scTPH”)  9 (AAD01923.1) Homo sapiens TPH2, insert(+6) 10 Homo sapiens TPH2, truncated ((45-471) + 20) 11 Homo sapiensTPH2, truncated (45-471) 12 Homo sapiens TPH2, truncated (147-460 + N-13 terminal methionine) GTP cyclohydrolase I Homo sapiens 14 (P30793)(EC 3.5.4.16) Mus musculus 15 (Q3U7P6) (GCH1) E. coli (FolE) 16 (P0A6T5)S. cerevisiae 17 (P51601) Bacillus subtilis 18 (G4EUF8) Streptomycesavermitilis 19 (Q82EE8) Salmonella typhii 20 (T2PZ12) pterin-4-alpha-Chromobacterium violaceum 21 (Q7NVH7) carbinolamine Pseudomonasaeruginosa 22 (P43335) dehydratase (EC Bacillus cereus var. anthracis 23(D8GWB2) 4.2.1.96) (PCD) Lactobacillus ruminis ATCC 25644 25 (E7FT68)Rhodobacteraceae bacterium HTCC2083 26 (B6B2H7) Homo sapiens 27 (P61457)5HTP decarboxylase Acidobacterium capsulatum 28 (WP_015898075.1) (EC4.1.1.28) Rattus norwegicus 29 (XP_006251536.1) (ADDC) Sus scrofa 30(NP_999019.1) Homo sapiens 31 (P20711-1, v2) Capsicum annuum 32(NP_001312016.1) Drosophila caribiana 33 (AAM80956.1) Maricaulis maris(strain MCS10) 34 (ABI65701.1) Oryza sativa subsp. Japonica 35(XP_015648768.1) Pseudomonas putida S16 36 (WP_013972057.1) Catharanthusroseus 37 (P17770-1, v1) serotonin Chlamydomonas reinhardtii 38(BAH10512.1) acetyltransferase (EC Bos Taurus 39 (DAA18183.1) 2.3.1.87or 2.3.1.5) Bos Taurus A55P 40 (AANAT) Gallus gallus 41 (NP_990489.1)Homo sapiens 42 (NP_001079.1) Mus musculus 43 (XP_011246971.1)Oryctolagus cuniculus 44 (XP 008249128.1) Ovis aries 45 (NP_001009461.1)acetylserotonin O- Oryza sativa 46 (XP 015610997.1) methyltransferaseHomo sapiens 47 (P46597-1, v1) (EC 2.1.1.4) (ASMT) Bos Taurus 48(P10950-1, v2) Rattus norvegicus 49 (NP_653360.2) Gallus gallus 50(NP_990674.1) Macaca mulatta 51 (NP_001028112.1) Ocimum basilicumQ9XGV9-1, v1 Takifugu rubripes (XP_011609423.1) Elephantulus edwardii(XP_006902482.1) Chromobacterium violaceum (WP_011135808.1)Desulfotomaculum kuznetsovii DSM 6115 (YP_004515712.1) Xenopus(Silurana) tropicalis (NP_001011409.1) Pseudomonas fluorescens(WP_019095725.1) Candidatus Solibacter usitatus (WP_011682595.1)Fenneropenaeus chinensis (AAZ66373.1) Arabidopsis thaliana (NP_200227.1)Tyrosine hydroxylase Rattus norwegicus 52 (NP_036872.1) (TH) (EC1.14.16.2) Homo sapiens 54 (P07101) Mus musculus 55 (P24529) Bos taurus56 (P17289) Gallus gallus 57 (Q9PU40) Phenylalanine Chromobacteriumviolaceum 58 (P30967) hydroxylase (PheH) Xanthomonas campestris pv.Viticola 59 (A0A077SF23) (EC 1.14.16.1, EC Pseudomonas aeruginosa 60(P43334) 1.14.16.7) Pseudomonas putida 61 (Q6EMJ5) Homo sapiens 62(P00439) Mus musculus 63 (P16331) Streptomyces coeruleorubidus 64(F5BFC8)

Variants or homologs of any one or more of the enzymes and otherproteins listed in Table 1, having the referenced activity and asequence identity of at least 30%, such as at least 50%, such as atleast 60%, such as at least 70%, such as at least 80%, such as at least90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98%, such asat least 99%, over at least the catalytically active portion, optionallyover the full length, of the reference amino acid sequence in its matureform, excluding signal peptide sequences and the like, are alsocontemplated. The variant or homolog may comprise, for example, 2, 3, 4,5 or more, such as 10 or more, amino acid substitutions, insertions ordeletions as compared to the reference amino acid sequence in its matureform. In particular conservative substitutions are considered. These aretypically within the group of basic amino acids (arginine, lysine andhistidine), acidic amino acids (glutamic acid and aspartic acid), polaramino acids (glutamine and asparagine), hydrophobic amino acids(leucine, isoleucine and valine), aromatic amino acids (phenylalanine,tryptophan and tyrosine), and small amino acids (glycine, alanine,serine, threonine and methionine). Amino acid substitutions which do notgenerally alter specific activity are known in the art and aredescribed, for example, by H. Neurath and R. L. Hill, 1979, In: TheProteins, Academic Press, New York. The most commonly occurringexchanges are Ala to Ser, Val to lie, Asp to Glu, Thr to Ser, Ala toGly, Ala to Thr, Ser to Asn, Ala to Val, Ser to Gly, Tyr to Phe, Ala toPro, Lys to Arg, Asp to Asn, Leu to lie, Leu to Val, Ala to Glu, and Aspto Gly. Homologs, such as orthologs or paralogs, having the desiredactivity can be identified in the same or a related animal or microbialspecies using the reference sequences provided and appropriate activitytesting. Specific enzyme variants are exemplified herein.

In a particularly preferred embodiment, the variant of an amino acidsequence in Table 1 has an X2K amino acid substitution, where “X”represents the amino acid adjacent to the N-terminal methionine residue.

The recombinant host cell is typically prepared by introducing,typically via transformation, one or more vectors as described herein,using standard methods known in the art (see, e.g., Sambrook et al.,2012, supra). The introduction of a vector into a bacterial host cellmay, for instance, be effected by protoplast transformation (see, e.g.,Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), usingcompetent cells (see, e.g., Young and Spizizen, 1961, Journal ofBacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journalof Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawaand Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g.,Koehler and Thome, 1987, Journal of Bacteriology 169: 5771-5278).

As described above, the vector, once introduced, may be maintained as achromosomal integrant or as a self-replicating extra-chromosomal vector.

Preferably, for transformation of an E. coli or other bacterial hostcell, the vectors are designed as follows: A lac promoter is used tocontrol the expressions of a gene or an artificial operon containing upto three genes connected with a linker sequence, in order to express thegenes at a suitable level so that the introduction of heterologousgenes/pathways do not overdraw substrates or energy in the host cell. Inone particular embodiment, the recombinant microbial cell, preferably abacterial cell, is transformed according to a strategy outlined in theExamples.

Preferably, for transformation of a yeast host cell such as S.cerevisiae, the heterologous genes are integrated onto chromosome usinga homologous recombination based method (Mikkelsen et al., 2012). Ascompared with gene expression based on plasmids, the chromosomalintegrated genes can be expressed with higher fidelity and resulted inbetter protein translation, in particular for multiple geneco-expression systems.

The transformation can be confirmed using methods well known in the art.Such methods include, for example, nucleic acid analysis such asNorthern blots or polymerase chain reaction (PCR) amplification of mRNA,or immunoblotting for expression of gene products, or other suitableanalytical methods to test the expression of an introduced nucleic acidsequence or its corresponding gene product, including those referred toabove and relating to measurement of 5HTP production. Expression levelscan further be optimized to obtain sufficient expression using methodswell known in the art and as disclosed herein.

Tryptophan, tyrosine and phenylalanine production takes place in allknown microorganisms by a single metabolic pathway (Somerville, R. L.,Herrmann, R. M., 1983, Amino acids, Biosynthesis and Genetic Regulation,Addison-Wesley Publishing Company, U.S.A.: 301-322 and 351-378; Aida etal., 1986, Bio-technology of amino acid production, progress inindustrial microbiology, Vol. 24, Elsevier Science Publishers,Amsterdam: 188-206). The recombinant microbial cell of the invention canthus be prepared from any microbial host cell, using recombinanttechniques well known in the art (see, e.g., Sambrook et al., 2012,supra, and Ausubel et al. (1991), supra. Preferably, the host cell istryptophan, tyrosine and/or phenylalanine autotrophic (i.e., capable ofendogenous biosynthesis of the substrate of the oxidation reaction ofinterest), grows on synthetic medium with suitable carbon sources, andexpresses a suitable RNA polymerase (such as, e.g., T7 polymerase).

GTP cyclohydrolase I (such as, e.g. FolE)-catalyzed pterin biosynthesistakes place in many organisms including both prokaryotes and eukaryotes.The recombinant cell of the invention can thus be prepared from anyhosts, using recombinant techniques well known in the art (see, e.g.,Sambrook et al., 2012, supra, and Ausubel et al. (1991), supra.Preferably, the host cell is capable of one, more or all oftetrahydrofolate, tetrahydrobiopterin, preQ₀, drosopterin,aurodrosopterin or tetrahydromonapterin biosynthesis (see FIG. 1). So,for example, in one embodiment, the microbial host cell is an E. colicell comprising the endogenous enzymes folE, folX, P-ase, and folM,optionally upregulated or expressed from one or more vectors.

For embodiments where the monooxygenase is a TPH and the desiredend-product is serotonin, N-acetylserotonin or melatonin, therecombinant host cell is typically capable of biosynthesizing and/orregenerating the cofactors used by the enzymes in the melatoninbiosynthesis pathway. In particular, the recombinant host cell ispreferably capable of biosynthesizing, regenerating, or bio-synthesizingand regenerating, one or more cofactors for TPH, AANAT and ASMT. Mosttypes of host cells (e.g., mammalian host cells, yeast host cells suchas S. cerevisiae, bacteria such as E. coli, etc.) are capable ofproducing and regenerating acetyl-CoA and SAM; the cofactors for AANATand ASMT, respectively. AcCoA serves as a metabolic cofactor in theAANAT reaction, but is also part of other, endogenous pathways in, e.g.,microbial cells.

SAM is a principal methyl donor in various intracellulartransmethylation reactions. It is synthesized in the cell through SAMsynthetase from methionine and ATP, and natively generated through theSAM cycle, which consists of a methyl transferase, anS-adenosyl-L-homocysteine hydrolase, a folate transferase, and anS-adenosyl-methionine synthetase (Lee et al., Korean J. Chem. Eng. 2010,27, 587-589). Accordingly, in the ASMT-catalyzed, last reaction in theproduction of melatonin from L-tryptophan, N-acetylserotonin and SAM areconverted to melatonin and SAH. SAH can then be recycled back to SAM viathe SAM-cycle in microbial cells where the S-adenosyl-L-methionine cycleis native (or exogenously added) and constitutively expressed, such as,e.g., in E. coli. The enzymes of such native pathways can also, inneeded, be upregulated or expressed from an exogenously introducedvector, using well-known recombinant techniques known in text booksreferenced elsewhere herein. Non-limiting and exemplary nucleic acidsencoding enzymes of the SAM cycle for use in aspects and embodiments ofthe present invention include those shown in Table 1 of WO 2015/032911A1, which is hereby specifically incorporated by reference, includingthe actual amino acid sequences referred to in the table as SEQ IDnumbers.

The microbial host cell for use in the present invention is typicallyunicellular and can be, for example, a bacterial cell, a yeast hostcell, a filamentous fungal cell, or an algeal cell. Examples of suitablehost cell genera include, but are not limited to, Acinetobacter,Agrobacterium, Alcaligenes, Anabaena, Aspergillus, Bacillus,Bifidobacterium, Brevibacterium, Candida, Chlorobium, Chromatium,Corynebacteria, Cytophaga, Deinococcus, Enterococcus, Erwinia,Erythrobacter, Escherichia, Flavobacterium, Hansenula, Klebsiella,Lactobacillus, Methanobacterium, Methylobacter, Methylococcus,Methylocystis, Methylomicrobium, Methylomonas, Methylosinus,Mycobacterium, Myxococcus, Pantoea, Phaffia, Pichia, Pseudomonas,Rhodobacter, Rhodococcus, Saccharomyces, Salmonella, Sphingomonas,Streptococcus, Streptomyces, Synechococcus, Synechocystis, Thiobacillus,Trichoderma, Yarrowia and Zymomonas.

In one embodiment, the host cell is bacterial cell, e.g., an Escherichiacell such as an Escherichia coli cell; a Bacillus cell such as aBacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, or a Bacillusthuringiensis cell; or a Streptomyces cell such as a Streptomyceslividans or Streptomyces murinus cell. In a particular embodiment, thehost cell is an E. coli cell. In another particular embodiment, the hostcell is of an E. coli strain selected from the group consisting ofK12.DH1 (Proc. Natl. Acad. Sci. USA, volume 60, 160 (1968)), JM101,JM103 (Nucleic Acids Research (1981), 9, 309), JA221 (J. Mol. Biol.(1978), 120, 517), HB101 (J. Mol. Biol. (1969), 41, 459) and C600(Genetics, (1954), 39, 440).

In one embodiment, the host cell is a fungal cell, such as, e.g., ayeast cell. Exemplary yeast cells include Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces and Yarrowiacells. In a particular embodiment, the host cell is an S. cerevisiaecell. In another particular embodiment, the host cell is of an S.cerevisie strain selected from the group consisting of S. cerevisiaeKA31, AH22, AH22R-, NA87-11A, DKD-5D and 20B-12, S. pombe NCYC1913 andNCYC2036 and Pichia pastoris KM71. The recombinant microbial cell of anyone of the aspects or embodiments herein is typically a bacterial cell,a yeast cell, a filamentous fungal cell, or an algal cell. In oneembodiment, the recombinant microbial cell is a cell of the Escherichiagenus, such as an Escherichia coli cell. In one embodiment, therecombinant microbial is derived from a Saccharomyces, aSchizosaccharomyces, a Corynebacterium, a Bacillus or a Streptomycescell.

Production of Oxidation Products and Downstream Products:

The invention also provides a method of producing various oxidationproducts, e.g., of aromatic amino acids, as well as various otherproducts where such an oxidation product is an intermediate in abiosynthetic pathway. Examples of products include, but are not limitedto, 5HTP, L-DOPA, tyrosine (e.g., m-tyrosine), hydroxytyrosol,melatonin, serotonin and/or N-acetyl-serotonin, comprising culturing therecombinant microbial cell of any preceding aspect or embodiment in amedium comprising a carbon source. The desired compound can thenoptionally be isolated or retrieved from the medium, and optionallyfurther purified. Importantly, using a recombinant microbial cellaccording to the invention, the method can be carried out without addingmonooxygenase substrates such as L-tryptophan, L-tyrosine orL-phenylalanine, or monooxygenase cofactor such as THB, or both, to themedium.

Also provided is a method of preparing a composition comprising one ormore compounds selected from 5HTP, L-DOPA, tyrosine, hydroxytyrosol,melatonin, serotonin and/or N-acetyl-serotonin, comprising culturing therecombinant microbial cell of any preceding aspect or embodiment,isolating and purifying the compound(s), and adding any excipients toobtain the composition.

Suitable carbon sources include carbohydrates such as monosaccharides,oligosaccharides and polysaccharides. As used herein, “monosaccharide”denotes a single unit of the general chemical formula Cx(H2O)y, withoutglycosidic connection to other such units, and includes glucose,fructose, xylose, arabinose, galactose and mannose. “Oligosaccharides”are compounds in which monosaccharide units are joined by glycosidiclinkages, and include sucrose and lactose. According to the number ofunits, oligosaccharides are called disaccharides, trisaccharides,tetrasaccharides, pentasaccharides etc. The borderline withpolysaccharides cannot be drawn strictly; however the term“oligosaccharide” is commonly used to refer to a defined structure asopposed to a polymer of unspecified length or a homologous mixture.“Polysaccharides” is the name given to a macromolecule consisting of alarge number of monosaccharide residues joined to each other byglycosidic linkages, and includes starch, lignocellulose, cellulose,hemicellulose, glycogen, xylan, glucuronoxylan, arabinoxylan,arabinogalactan, glucomannan, xyloglucan, and galactomannan. Othersuitable carbon sources include acetate, glycerol, pyruvate andgluconate. In one embodiment, the carbon source is selected from thegroup consisting of glucose, fructose, sucrose, xylose, mannose,galactose, rhamnose, arabinose, fatty acids, glycerine, glycerol,acetate, pyruvate, gluconate, starch, glycogen, amylopectin, amylose,cellulose, cellulose acetate, cellulose nitrate, hemicellulose, xylan,glucuronoxylan, arabinoxylan, glucomannan, xyloglucan, lignin, andlignocellulose. In one embodiment, the carbon source comprises one ormore of lignocellulose and glycerol. In one embodiment, the carbonsource is a simple carbon source such as glucose, xylose, fructose,arabinose, galactose, mannose, glycerol, acetate, or a mixture of anythereof.

The culture conditions are adapted to the recombinant microbial hostcell, and can be optimized to maximize production or melatonin oranother desired compound by varying culture conditions and mediacomponents as is well-known in the art.

For a recombinant Escherichia coli cell, exemplary media include LBmedium and M9 medium (Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, 1972),optionally supplemented with one or more amino acids. When an induciblepromoter is used, the inductor can also be added to the medium. Examplesinclude the lac promoter, which can be activated by addingisopropyl-beta-thiogalacto-pyranoside (IPTG) and the GAL/BAD promoter,in which case galactose/arabinose can be added. The culturing can becarried out a temperature of about 10 to 40° C. for about 3 to 72 hours,if desired, with aeration or stirring.

For a recombinant Bacillus cell, culturing can be carried out in a knownmedium at about 30 to 40° C. for about 6 to 40 hours, if desired withaeration and stirring. With regard to the medium, known ones may beused. For example, pre-culture can be carried out in an LB medium andthen the main culture using an NU medium.

For a recombinant yeast cell, Burkholder minimum medium (Bostian, K. L.,et al. Proc. Natl. Acad. Sci. USA, volume 77, 4505 (1980)), SD mediumcontaining 0.5% of Casamino acid (Bitter, G. A., et al., Proc. Natl.Acad. Sci. USA, volume 81, 5330 (1984), and Delft medium (Verduyn etal., Yeast 1992, 8, 501-517) can be used. The pH is preferably adjustedto about 5-8. For example, a synthetic medium may contain, per litre:(NH4)2SO4, 5 g; KH2PO4, 3 g; MgSO4.7H2O, 0.5 g; EDTA, 15 mg; ZnSO4.7H2O,4.5 mg; CoCl2.6H2O, 0.3 mg; MnCl2.4H20, 1 mg; CuSO4 5H2O, 0.3 mg;CaCl2.2H2O, 4.5 mg; FeSO4.7H2O, 3 mg; NaMoO4.2H2O, 0.4 mg; H3BO3, 1mg-KI, 0.1 mg; and 0.025 ml silicone antifoam (BDH). Filter-sterilizedvitamins can be added after heat sterilization (120° C.), to finalconcentrations per litre of: biotin, 0.05 mg; calcium pantothenate, 1mg; nicotinic acid, 1 mg; inositol, 25 mg; thiamine HCl, 1 mg;pyridoxine HCl, 1 mg; and para-aminobenzoic acid, 0.2 mg. The medium canthen be adjusted to pH6 with KOH. Culturing is preferably carried out atabout 20 to about 40° C., for about 24 to 84 hours, if desired withaeration or stirring.

In one embodiment, no L-tryptophan is added to the medium. In anotherembodiment, no L-tryptophan or THB is added to the medium, so that theproduction of melatonin or its precursors or related compounds rely onendogenously biosynthesized substrates.

Using the method for producing 5HTP, L-DOPA, tyrosine, m-tyrosine,hydroxytyrosol, melatonin, serotonin or N-acetyl-serotonin according tothe invention, a yield of at least about 0.5%, such as at least about1%, such as at least 5%, such as at least 10%, such as at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80% or at least 90% of the theoretically possible yield can beobtained from a suitable carbon source, such as glucose.

Isolation of 5HTP, L-DOPA, tyrosine, m-tyrosine, hydroxytyrosol,melatonin, N-acetylserotonin or serotonin from the cell culture can beachieved, e.g., by separating the compound from the cells using amembrane, using, for example, centrifugation or filtration methods. Theproduct-containing supernatant is then collected. Further purificationof the desired compound can then be carried out using known methods,such as, e.g., salting out and solvent precipitation;molecular-weight-based separation methods such as dialysis,ultrafiltration, and gel filtration; charge-based separation methodssuch as ion-exchange chromatography; and methods based on differences inhydrophobicity, such as reversed-phase HPLC; and the like. In oneembodiment, ion-exchange chromatography is used for purification ofserotonin. An exemplary method for serotonin purification usingcation-exchange chromatography is described in Chilcote (1974) (ClinChem 20(4):421-423). In one embodiment, reverse-phase chromatography isused for separation and/or purification of serotonin, N-acetylserotonin,or melatonin. An exemplary method for purification of these indolaminesusing reversed-phase chromatography is described in Harumi et al.,(1996) (J Chromatogr B 675:152-156).

Once a sufficiently pure preparation has been achieved, suitableexcipients, stabilizers can optionally be added and the resultingpreparation incorporated in a composition for use in preparing a productsuch as, e.g., a dietary supplement, a pharmaceutical, a cosmeceutical,or a nutraceutical. For a dietary supplement comprising melatonin, eachserving can contain, e.g., from about 0.01 mg to about 100 mg melatonin,such as from about 0.1 mg to about 10 mg, or about 1-5 mg, such as 2-3mg. Emulsifiers may be added for stability of the final product.Examples of suitable emulsifiers include, but are not limited to,lecithin (e.g., from egg or soy), and/or mono- and di-glycerides. Otheremulsifiers are readily apparent to the skilled artisan and selection ofsuitable emulsifier(s) will depend, in part, upon the formulation andfinal product. Preservatives may also be added to the nutritionalsupplement to extend product shelf life. Preferably, preservatives suchas potassium sorbate, sodium sorbate, potassium benzoate, sodiumbenzoate or calcium disodium EDTA are used.

Example 1

Media and Growth Conditions

All strains were maintained at 37° C. in LB (Lennox) Broth(Sigma-Aldrich), 2×YT or M9 minimum media containing 1×M9 minimal salts(BD Difco™), 2 mM MgSO₄, 100 μM CaCl₂, 500-fold diluted trace minerals(10 g/l FeCl₃.6H₂O, 2 g/l ZnSO₄.7H₂O, 0.4 g/l CuCl₂-2H₂O, 1 g/lMnSO₄—H₂O, 0.6 g/l CoCl₂-6H₂O, and 1.6 mM EDTA, pH 8.0), lx ATCC®Vitamin Supplement (ATCC MD-VS™), and 0.2% glucose (w/v). Unless statedotherwise, required supplementations were carried out as following:L-phenylalanine at 50 mg/l, L-tyrosine at 30 mg/1, folates at 10 mg/l,chloramphenicol at 25 mg/l, kanamycin at 25 mg/l, and spectinomycin at50 mg/l.

Plasmids

The pPheH plasmid carries a codon-optimized pheH from C. violaceum. ThepTHBsc plasmid carries the pcd, qDPR, pts and spr genes. The pcd andqDPR genes are from C. violaceum while the pts and spr genes are fromRattus norvegicus. The pHM1 plasmid was derived from pTHBscEv, anevolved pTHBsc plasmid isolated after PheH-dependent laboratoryevolution, containing the human TpH2 gene and a sacB gene, which causesplasmid instability upon exposure to sucrose. All synthetic genes wereoptimized for E. coli expression.

Metabolite Analysis by LC-MS

LC-MS data was collected on OrbiTrap Fusion High Resolution MassSpectrometer system coupled with an Ultimate 3000 UHPLC pump (Thermo,San Jose Ca). Samples were held in the autosampler at a temperature of10.0° C. during the analysis. 1 μL Injections of the sample were madeonto a Thermo HyperSil Gold PFP HPLC column, with a 3 um particle size,2.1 mm i.d. and 150 mm long. The column was held at a temperature of35.0° C. The solvent system used was Solvent A “Water with 0.1% formicacid” and Solvent B “Acetonitrile with 0.1% formic”. The Flow Rate was1.000 ml/min with an Initial Solvent composition of % A=95, % B=5 helduntil 0.50 min, the solvent composition was then changed following aLinear Gradient until it reached % A=70.0 and % B=30.0 at 1.50 min. Thesolvent composition was then changed following a Linear Gradient untilit reached % A=5.0 and % B=95.0 at 2.00 min This was held until 2.50 minwhen the solvent was returned to the initial conditions and the columnwas re-equilibrated until 3.00 min. The first 0.25 min of the run wasdiverted to waste using the divert valve, following which the columneluent flowed directly into the Heated ESI probe of the MS which washeld at 325° C. and a voltage of 3500 V. Data was collected in positiveion mode over the mass range 50 to 1000 m/z at a resolution of 15.000.The other MS settings were as follows, Sheath Gas Flow Rate of 60 units,Cone Gas Flow Rate of 20 units Cone Temp was 275° C.

H₄-BPt Optimization by Phenylalanine-Hydroxylase Dependent LaboratoryEvolution

Patent applications PCT/EP2013/054019 and PCT/EP2014/068967 described amethod for the production of 5-hydroxytryptophan and melatonin byheterologous expression of human tryptophan hydroxylase (TpH) in E.coli. In addition to the desired biosynthetic genes,teterhydrobiopterine (H₄—BPt) synthesis and recycling pathwaysconsisting of four genes have to be introduced to accommodate TpHcofactor usage. Preliminary studies indicated that the rate-limitingstep was human TpH turnover, possibly due to limitations in cofactorsupply.

To overcome this challenge, a phenylalanine-hydroxylase dependentgrowth-coupled selection was designed. The key to this design was: 1) toconstruct an E. coli tyrosine auxotroph; 2) to introduce anH₄—BPt-dependent phenylalanine hydroxylase (PheH) that convertsphenylalanine to tyrosine. The chosen PheH was from Chromobacteriumviolaceum (SEQ ID NO:58) and the chosen background strain was HL1308.The E. coli HL1308 strain was constructed from JW2300 of the Keiocollection (Baba et al, 2006) with tyrA, recA and galE deletions furtherintroduced according to Datsenko and Wanner (2000) or by P1transduction. The HL1305 strain is genotypically similar to HL1308except the tyrA deletion. Deletion of tyrA, donated by ΔtyrA, resultedin a tyrosine auxotroph while ΔfolX was necessary to inactivate de novobiosynthesis of tetrahydromonapterin known to be an electron donor ofPheH from Pseudomonas aeruginosa (Pribat et al, 2010). The recA and galEgenes were removed respectively to minimize DNA recombination duringlaboratory evolution and to acquire a high DNA uptake efficiency.Constitutive expression and regulations of the heterologous genes wereenabled via a hybrid sequence of the native TyrR-repressible aroFpromoter sequence (Cobbett, 1988) and the translation leader sequence ofthe rpsA gene (Tchufistova et al, 2003).

Preliminary results confirmed HL1308 strain was unable to grow withouttyrosine supplementation. On the other hand, its tyrosine auxotrophy wasreverted upon transformation of the pTHBsc and pPheH plasmids. Thetransformed strain grew at an approximately half the rate of the controlstrain (HL1305) with phenylalanine supplementation; however, there wasno observable growth in the absence of phenylalanine.

Subsequently, the transformed strain was subjected to 280 generations ofmanual passage using shake flasks. The procedure was to inoculate 25 mlfresh phenylalanine supplemented M9 in a 250 ml flask to an initialdensity which was expected to reach an OD₆₀₀ of 0.2-0.4 after 24 hour ofincubation in an orbital 31 IDS shaker (Labnet) at 37° C. and 250 rpm.This serial transfer was repeated for 16 days. The initial phenylalanineconcentration was 50 mg/l and was lowered to 5 mg/l starting from day 7.Colonies were isolated at Day 6 and Day 16 and their growth rates weredetermined.

Adapted isolates from the high phenylalanine conditions displayed a widerange of growth response from barely growth to a growth rate comparableto HL1305 in the presence of 50 mg/I of phenylalanine. This result wasin contrast to the ones recovered from the low phenylalanine environmentwhereas all strains exhibited significant improvement in growth with amedian rate of 0.5±0.03 h⁻¹ and all were able to propagate in M9 withoutphenylalanine supplementation. The wider distribution in the growthrates of high-phenylalanine adapted isolates led us to hypothesize thatcertain strains in the population were evolved to become a tyrosinesecretor when an excess amount phenylalanine was present. We examinedthe hypothesis by feeding 0.5 g/l of phenylalanine to the best-grownstrain, THB402F3C9, and confirmed ˜0.1 g/l of tyrosine was secretedafter overnight growth.

Plasmids originated from adapted isolates were sequenced. It appearedthe pTHBsc plasmid from all isolates lacked the H₄—BPt biosynthesis andrecycling genes except pcd thus all were H₄—BPt null strains. Inaddition, there were neither mutations presented in the pheH and pcdgenes nor on the non-translational regions of the plasmids. Combiningboth evidences, it was reasoned E. coli cells must have repurposed itsown metabolism to fulfil PheH cofactor requirement during adaptation andpterine-4α-carbinolamine dehydratase encoded by pcd was the onlyadditional requirement.

A series of deletion mutants were made to elucidate possible native PheHcofactor in E. coli. Since folic acids and 6-carboxy-tetrahydropterin(H₄—CPt) share the same metabolic precursor as of H₄—BPt, it wasreasoned one of them or intermediates were activated as the electrondonor of PheH. To test this idea, their respective biosynthesis genes,queD and nudB, were deleted from the HL1308 ΔtyrR strain (HL1310). TheΔqueD strain (HL1312) exhibited no obvious growth defect upontransformation of pTHBscEv and pPheH (Table 2). On the other hand, theΔnudB strain (HL1314) exhibited severely impaired PheH-dependent growthwhile restoration of the tyrosine auxotrophy in HL1314 led to a strain(HL1314R) whose growth rate was comparable to control (Table 2).

Physiological characterizations indicated folic acids and theirintermediates must have contributed to the turnover of PheH in theevolved strains. The 6-hydroxymethyl-dihydropterin (6-CH₂OH—H₂Pt) is ametabolic intermediate of folic acids biosynthesis. Enzymaticcharacterizations have shown FolM reduces 6-CH₂OH—H₂Pt to6-hydroxymethyl-tetrahydropterin (6-CH₂OH—H₄Pt) in vitro (Pribat et al,2010) (FIG. 1). Due to its structural resemblance to H₄—BPt (FIG. 1insert), a ΔfolM strain was made but growth measurements showed mutantbehaved similar to HL1310 eliminating 6-CH₂OH—H₄Pt as a functionalcomplement (Table 2). This result was supported by the making of HL1330,a ΔpabA strain that is capable to synthesize all intermediates of thefolic acids biosynthesis pathway from dihydroneopterin triphosphate(H₂—NPtP₃) to dihydropteroate (H₂—Pte) except folic acids (FIG. 1).Growth results showed folates supplementation could rescue JW3323(BW25113 ΔpabA) (Baba et al, 2006) in M9 but not PheH-dependent growthof HL1330. These results have led us to conclude the tetrahydrofolicacids were the acting donor of PheH in the evolved strain.

TABLE 2 Growth measurements of various mutants ^(T) growth rate,inoculation OD₆₀₀ after Strains Genotype h⁻¹ OD₆₀₀ 8 h comments HL1305BW25113 ΔfolX 0.59 0.035 0.593 control ΔrecA ΔgalE strain HL1308¹BW25113 ΔfolX 0.244 0.025 0.178 ΔtyrA ΔrecA ΔgalE HL1310¹ HL1308 ΔtyrR0.33 0.023 0.382 HL1312¹ HL1310 ΔqueD 0.27 0.024 0.155 HL1314¹ HL1310ΔnudB no 0.02 0.051 growth HL1314R HL1314 tyrA⁺ 0.58 0.02 0.6 HL1326¹HL1310 ΔfolM 0.3 0.027 0.289 JW3323^(LI) BW25113 ΔpabA 0.4 0.036 0.488Keio collection HL1330^(1, LI) HL1310 ΔpabA no 0.022 0.035 growthTHB402F3C9 HL1308 ΔrpsA² 0.58 0.027 0.61 evolved rpoB:E546K strainrpoC:I1357S ^(T) Growth measurements were performed in M9 minimum mediawith 50 mg/l phenylalanine. ^(LI)Folic acids at 10 mg/1 were added.¹Cells were transformed with pTHBscEv and pPheH. ²The rpsA gene isinactivated by a frameshift deletion at the 9th nucleotide after the ATGstart codon.Tryptophan-Hydroxylase Dependent Laboratory Evolution

Based on the phenylalanine-hydroxylase study, we hypothesized that itwas possible to use native E. coli compounds (e.g. folate species) tosupport AAH activity with functional expression of the pcd gene beingthe only requirement. Since it was known that TpH is capable ofphenylalanine turnover to form tyrosine in vitro, thus it is plausibleto adjust TpH by evolution using the same principle.

In the subsequent evolution, TpH replaced the PheH in the previousevolution. This was done by inserting a Ptrc (Trc promoter)-regulatedhuman TpH gene (encoding SEQ ID NO: 13) onto pTHBscEv along with acounter-selectable sacB gene. The resulting plasmid, pHM1, wasintroduced to a tyrosine auxotroph HM30 strain, which was derived fromJW2581 of the KEIO collection but with the kanamycine-resistant markerat the tyrA locus removed. The final transformed strain was referred asHM35. The HM35 cells were then growth-adapted for high growth rate inthe presence of phenylalanine.

At the end of experiment, 94 isolates were picked and were grown in M9medium supplemented with 200 mg/I of tryptophan and 76 mg/l of tyrosine.The amount of 5HTP formed in the exo-metabolom was measured, it wasobserved some isolates were able to accumulate more than 10 mg/I of 5HTPand this was ˜10-fold more than their parent strain (HM35).

A total of 7 high 5HTP-producers were subjected to total DNA sequenceanalysis. Three mutations were found common in all strains: FolE(T198I), YnbB (V197A) and TpH (E2K, in SEQ ID NO:13). Both FolE (T198I)and YnbB (V197A) mutations were genomic changes while the TpH (E2K)mutation occurred on the pHM plasmid. Further analyses indicated thatthe YnbB (V197A) mutation might not be any functional importance sinceit was also found in the parent HM35 strain. However, without beinglimited to theory, the T198I change in FolE may have resulted inincreased supply of a TpH cofactor as FolE encoding for GTPcyclohydrolase I is the first enzyme leading to tetrahydrofolates,tetrahydromonapterin and others which may act as TpH cofactors in E.coli. This is a particularly interesting result, since preliminarystudies have indicated that the catalytic effectiveness of FolE cannotbe easily increased by gene overexpression. Furthermore, a proteomicstudy revealed that the E2K mutation resulted in ˜20 fold increase inTpH abundance in contrast to the wild-type form.

Example 2

Directed TpH Engineering

It was found that Homo sapiens TpH2, i.e., the fragment set forth as SEQID NO:13; hsTpH2, was sensitive to p-chlorophenylalanine. However,mutations at residues N97 and/or P99 were found to confer resistance top-chlorophenylalanine and to exhibit improved 5HTP biosynthesis aftergrowing cells in the presence of 100 mg/l of tryptophan overnight at 37°C. A further, saturated mutagenesis, study found that isoleucine (I) wasa beneficial amino acid change at residue N97, while cysteine (C),aspartic acid (D), leucine (L) and glutamine (Q) were shown to bebeneficial at residue P99. In particular, the combined changes N97I/P99Din hsTpH2 showed a >15% increase in 5HTP production in the presence of100 mg/l tryptophan and the combined changes N97I/P99C in hsTpH2 showeda >25% increase in 5HTP biosynthesis, over the parent TPH2 sequence (SEQID NO:13) after acquiring the E2K mutation.

Example 3

Effectiveness of Newly Evolved TpH Background Strain Using Schistosomamansoni TpH

One of the 7 evolved high 5HTP-producers was selected to furtherevaluate if the mutations identified were only specifically beneficialto hsTpH2 or could be widely applicable to others. The chosen evolvedstrain was first cured to lose the evolution plasmid (e.g. the hsTpHgene) and this was immediately followed by re-introducing the E. colityrA gene. Upon restoration of the strain's tyrosine auxotrophy, theresulting strain was transformed with pHM2, which is identical to pHMused in the earlier evolution study except that the hsTpH gene wasreplaced with a Schistosoma mansoni TpH gene (SEQ ID NO:9). The 5HTPproduction of the resulting strain was compared to a wild-type straincarrying pHM2 in the presence of 100 mg/l tryptophan. Results showed thewild-type transformants could only produce ˜0.05 mg/l 5HTP while thenewly evolved background strain transformants accumulated >20 mg/l.These production results demonstrated that the mutations acquired in theevolved background strain were not only beneficial to hsTpH but also toother TpHs; possibly applicable also to other aromatic amino acidhydroxylases (e.g. tyrosine hydroxylase).

Example 4

Effect of FolE Mutation

This Example shows that the FolE(T198I) mutation is a beneficialmutation towards TpH functionality.

It was assumed the FolE mutation was the main contributor on improvingTpH turnover. To challenge this assumption, the FolE(T198I) mutation wasintroduced into the wild-type BW25113 strain by site-directedmutagenesis using CRISPR. All strains were transformed with a plasmidcarrying the human TPH and C. violaceum PCD genes. The transformed cellswere grown in M9 medium containing 100 mg/l tryptophan overnight andextracellular metabolites were subjected to analytical measurements. Theresults are summarized in Table 3.

TABLE 3 Importance of FolE(T198I) on in vivo hsTpH¹ turnover in thepresence of 100 mg/1 of tryptophan Genotype 5HTP (mg/l) Indole (mg/l)5-hydroxyindole (mg/l) wild-type n.d. 85 ± 5 n.d. ΔTnaA 40 ± 2 n.d. n.d.FolE(T198I) n.d. n.d. n.d. ΔTnaA 129 ± 30 n.d. n.d. FolE(T198I) ¹hsTpHcarries E2K, N97I and P99C mutations in SEQ ID NO: 13 n.d.: not detected

As shown in Table 3, wild-type hsTpH expressing cells did not produce5HTP under tryptophan feeding conditions. However, 5HTP production couldbe observed with TnaA deletion, which prevented tryptophan and 5HTPdegradation. Further 3-folder enhancement on 5HTP production wasachieved with FolE(T198I) on ΔTnaA background. Accordingly, the FolEchange is a beneficial mutation towards TpH functionality.

Example 5

Tyrosine Hydroxylase Activity

This Example shows that the FolE(T198I) mutation is a beneficialmutation also for supporting tyrosine hydroxylase functionality.

Since both tyrosine hydroxylase and tryptophan hydroxylase belong to thesame class of enzyme (i.e., utilizing the same cofactor for oxidation),it was also expected FolE(T198I) would benefit tyrosine hydroxylaseactivity in terms of converting L-tyrosine toL-3,4-dihydroxyphenylalaine (L-DOPA). Both wild type E. coli andFolE(198I) cells were transformed with a plasmid carrying either thewild-type or truncated rat TH gene expressed from a trc promoter.Additionally, the proline residue of the 2^(nd) amino acid of thetruncated gene was replaced with lysine. The transformed cells were grewin M9 containing 100 mg/I tyrosine overnight and extracellularmetabolites were subjected to analytical measurements. As summarized inTable 4, it was shown that the presence of FolE mutation enhanced L-DOPAformation by about 3-fold when compared to the ones lacking themutation. Furthermore, it was also shown that removal of the N-terminalmammalian signalling amino acids and substituting the 2^(nd) amino acidfrom proline to lysine additionally enhanced L-DOPA formation about4-fold. It was therefore a total of c.a. 10-fold improvement achieved bycombining all the modifications. Without being limited to theory, it isbelieved that the FolE mutation could benefit other enzymes of the sameclass, including phenylalanine hydroxylase (EC 1.14.16.1), phenylalanine3-hydroxylase (EC 1.14.16.7), tyrosine 3-hydroxylase (EC 1.14.16.2),anthranilate 3-monooxygenase (EC 1.14.16.3), mandelate 4-monooxygenase(EC 1.14.16.6) and Alkylglycerol monooxygenase (EC 1.14.16.5).

TABLE 4 Importance of FolE(T198I) on in vivo tyrosine hydroxylaseturnover in the presence of 100 mg/l of tyrosineL-3,4-dihydroxyphenylalanine Tyrosine hydroxylase¹ FolE(T198I) (LDOPA)(mg/g dry cell weight) wild-type² No 2.88 ± 0.1 wild-type² Yes  7.25 ±0.69 P2K Δ(3-157) No 10.62 ± 0.71 P2K Δ(3-157) Yes  30.3 ± 1.43¹tyrosine hydroxylase from Rattus norvegicus ²NCBI accession number:NP_036872.1

Example 6

Comparing FolE Overexpression with FolE Mutation

This Example shows that overexpression of the wild-type folE gene didnot achieve an FolE(T198I) effect.

Gene overexpression has often been used to enhance in vivo enzymaticturnover. As determined in Example 1, TPH-dependent cell growth basingon phenylalanine to tyrosine conversion was an effective way to reflectTPH turnover and its associated networked genes, including its cofactorsupply. Therefore, a TPH-dependent growth experiment was set up tocompare the effectiveness of the FolE mutation and gene overexpression.Overexpression of the folE gene was achieved by introducing a folEbearing plasmid with its native E. coli promoter intact. TheTPH-dependent HM30 cells were transformed with two plasmids: a TPH/PCDbearing plasmid and the concerning folE bearing plasmid. The transformedstrains were grown in M9 in the presence of 100 mg/l phenylalanine at37° C. along with control strains carrying either wild-type chromosomalFolE or FolE(T198I) in additional to TPH/PCD expression from a plasmid.Cell growth was measured at regular intervals.

The results are summarized in FIG. 2. As shown, strains carrying achromosomal FolE mutation (gFolE*) exhibited rapid TPH-dependent growthduring the time course of this experiment. This was in contrast to thenative chromosomal variant (gFolE), which barely grew. Although it wasinteresting to see cells with overexpression of wild-type FolE(gFolE+pFolE) exhibited elevated cell growth yet its growth was not asrapid as the single copy chromosomal mutant. Furthermore, it wasobserved that cells carrying a hybrid of native and mutated FolE genes(gFolE+pFolE*) showed improved growth; however, its growth was not asgood as homogeneous chromosomal mutant (gFolE*). The reason was believedto be that since the T198 residue was located in a peptide segmentaffecting FolE oligomerization, a hybrid pool of the FolE proteins wouldultimately affect the polymerization of the FolE complex and hence itsoverall activity.

Example 7

Screen of FolE Library

This Example shows that the conformation of the FolE oligomerizationhelix (Ser207-Arg219) affects GTP cyclohydrolase I activity.

Structural analysis showed that the T198 residue is located in the4^(th) β-strand that goes in a close proximity to a α-helix defined bySer207 to Asn222, which can be involved in oligomerization of the FolEcomplex. Accordingly, T198 may play a role in the FolE oligomerization.Specifically, its mutation would affect the hydrophobic interaction withLeu215 and subsequently the positioning of Ser207-Arg219 helix andoverall polymerization of the FolE complex. It can therefore bepredicted that additional beneficial FolE mutations can be located inthe segments corresponding to residues D97-E112, residues K121-D130,residues N170-H180, residues S193-L200 and residues S207-N222.

A folE gene random library was constructed and was screened by growthaccording to Example 6. None-redundant growth-improved mutants and theircorresponding mutations are summarized in Table 5.

TABLE 5 Summary of beneficial FolE mutations identified by randommutagenesis Mutant growth Improvement Mutants FolE mutations rate, h⁻¹over wt HMP1058¹ Wild type 0.19 (1)   HMP1059² T198I 0.25 1.32 P1-01I67V, T117I, A125D, 0.32 1.68 H221R P1-02 V179A 0.32 1.68 P1-03 M99I0.36 1.89 P1-04 E62K, N170K, L215P 0.41 2.16 P1-05 V102M, L215P 0.351.84 P1-06 T198S 0.32 1.68 P1-07 Q157L, H212R 0.36 1.89 P1-08 V28L,L215P, N222I 0.4 2.11 P1-10 F214S 0.32 1.68 P1-11 T108N, I133F, E213K0.35 1.84 P1-12 Q157L, H212R 0.37 1.95 P2-01 S5C, D57V, L215Q 0.36 1.89P2-02 H29Y, I75V, V179M 0.32 1.68 P2-03 A14V, E46D, M61I, D97V 0.36 1.89P2-04 V28A, G42D, E213K 0.4 2.11 P2-05 N52K, A68S, S207R 0.36 1.89 P2-06L200P 0.36 1.89 P2-07 A41G, K129N, I133F 0.3 1.58 P2-09 S3L, K184R,S199Y 0.32 1.68 P2-11 H12R, N170D, G187S 0.39 2.05 ¹In Example 6illustrated as a mutant of gFolE + pFolE ²In Example 6 illustrated as amutant of gFolE + pFolE* .: cell growth was measured in M9 with 100 mg/lof phenylalanine at 37° C. overnight.

As shown in Table 5, mutants carrying E213K and L215P or L215Q showedthe most significant growth improvement following by D97V and M99I ofthe D97-E112 segment, N170K and N170D of the N170-H180 segment, andL200P, S207R and H212R of the S207-N222 segment.

Other mutations included V102M of the D97-E112 segment, A125D and K129Nof the K121-D130 segment, V179A and V179M of the N170-H180 segment,T198S and S199Y of S193-L200 segment, and F214S, H221R of the S207-N222segment. Overall, these results strongly suggest that mutations alteringFolE oligomerization can significantly alter the overall turnover rateof GTP cyclohydrolase I (FolE).

LIST OF REFERENCES

-   Lin et al., ACS synthetic biology 2014; 3:497-505-   Ehrenworth et al., ACS Synth Biol. 2015 Dec. 18; 4(12):1295-307.-   Yamamoto et al., Metab Eng 2003; 5:246-25-   Hara and Kino, AMB Express 2013; 3:70-   McKenzie G and Nancy LC (2006) BMC Microbiol 6:39-   Nar et al., PNAS USA 1995; 92:12120-5-   Rebelo et al., J. Mol. Biol. 2003; 326:503-516-   Pribat et al., J Bacteriol 2010a; 192(2):475-82-   Pribat et al., Plant Cell 2010b; 22(10):3410-22-   Baba et al., Mol Syst Biol 2006:2: 1-11-   Cobbett et al., Mol Microbiol 1988; 2: 377-383-   Datsenko and Wanner Proc Natl Acad Sci USA 2000; 97: 6640-6645-   Pribat et al., J Bacteriol 2010:192: 475-482-   Satoh et al., Metabolic engineering 2012; 14:603-610-   Tchufistova et al., Nucleic Acids Res 2003:31: 6996-7002-   WO 2013/127914 A1, WO 2013/127915 A1 and WO 2015/032911 A1 (Danmarks    Tekniske Universitet)-   US 2014/134689 AA (University of California)-   U.S. Pat. No. 7,807,421 (Asubio Pharma Co., Ltd)

The invention claimed is:
 1. A variant of E. coli GTP cyclohydrolase I(GCH1) having at least 94% sequence identity to native E. coli GCH1having the sequence of SEQ ID NO:16 and comprising one or moremutations, wherein, in an E. coli cell comprising apterin-4α-carbinolamine dehydratase (PCD) and at least one of atryptophan hydroxylase (TPH), a tyrosine hydroxylase (TH) and aphenylalanine hydroxylase (PheH), the variant provides for an increasedhydroxylation activity of at least one of the TPH, TH and PheH ascompared to native E. coli GCH1, wherein at least one of the one or moremutations is in an amino acid residue selected from the group consistingof D97, M99, T101, V102, A125, K129, N170, V179, T196, T198, S199, L200,S207, H212, E213, F214, L215 and H221, and wherein the mutation in N170is N170K, N170D or N170L; the mutation in T198 is T198I, T198V or T198L;the mutation in V179 is V179A; the mutation in H212 is H212R or H212K;and the mutation in H221 is H221R or H221K.
 2. The variant of claim 1,wherein the variant comprises a mutation selected from D97V, D97L, D97A,D97T, M99C, M99T, M99V, M99L, M99I, T101I, T101V, T101L, V102M, N170K,N170D, N170L, V179A, T1961, T196V, T196L, T198I, T198V, T198L, S199Y,S199F, L200P, L200C, L200S, L200A, S207R, S207K, S207M, H212R, H212K,E213K, E213R, F214A, F214G, F214S, L215P, L215Q, L215N, L215D, L215T,L215S, L215G, L215A, L215C, L215F, L215M, H221R and H221K, or acombination thereof.
 3. The variant of claim 1, comprising a mutationselected from T198I, F214S, V179A, M99I, L200P and L215P.
 4. A nucleicacid sequence encoding the variant of E. coli GCH1 of claim
 1. 5. Arecombinant microbial cell comprising the variant of claim
 1. 6. Therecombinant microbial cell of claim 5, further comprising nucleic acidsequences encoding a PCD and a monooxygenase.
 7. The recombinantmicrobial cell of claim 6, wherein (a) The recombinant microbial cell ofclaim 6, wherein (a) the PCD is derived from Chromobacterium violecum,Homo sapiens, Pseudomonas aeruginosa and Rattus norvegicus; or afunctionally active variant thereof; (b) the monooxygenase is a TPHderived from a Schistosoma mansoni, Homo sapiens, Gallus gallus, Bostaurus, Sus scrofa, Equus caballus, Mus musculus and Oryctolaguscuniculus TPH; or a functionally active variant thereof; (c) themonooxygenase is a TH derived from Rattus norwegicus, Homo sapiens, Musmusculus, Bos taurus, Gallus gallus or a functionally active variantthereof; (d) the monooxygenase is a PheH derived from Chromobacteriumviolaceum, Xanthomonas campestris pv. Viticola, Pseudomonas aeruginosa,Pseudomonas putida, Homo sapiens, Mus musculus, Streptomycescoeruleorubidus or a functionally active variant thereof; or (e) acombination of (a) and (b), (a) and (c) or (a) and (d).
 8. Therecombinant microbial cell of claim 6, wherein each nucleic acidsequence is operably linked to an inducible, a regulated or aconstitutive promoter, and/or wherein at least the nucleic acid sequenceencoding the variant of E. coli GCH1 is chromosomally integrated.
 9. Therecombinant microbial cell of claim 6, which is derived from a bacterialcell, a yeast cell, a filamentous fungal cell, or an algal cell.
 10. Therecombinant microbial cell of claim 9, which is derived from anEscherichia, a Saccharomyces, a Schizosaccharomyces, a Corynebacterium,a Bacillus or a Streptomyces cell.
 11. The recombinant microbial cell ofclaim 6, further comprising (a) a nucleic acid sequence encoding a 5HTPdecarboxylase (ADDC); (b) nucleic acid sequences encoding an ADDC and aserotonin acetyltransferase (AANAT); (c) nucleic acid sequences encodingan ADDC, an AANAT, and an acetylserotonin O-methyltransferase (ASMT); or(d) nucleic acid sequences encoding a dopa decarboxylase, a tyramineoxidase and an alcohol dehydrogenase.
 12. A method of producing one ormore oxidation products of an aromatic amino acid, comprising culturingthe recombinant microbial cell of claim 6 in a medium comprising acarbon source, and, optionally, isolating the oxidation product.
 13. Themethod of claim 12, wherein the oxidation product comprises at least oneof 5HTP, L-DOPA, tyrosine, m-tyrosine, hydroxytyrosol, serotonin andmelatonin.
 14. A variant of claim 1, E. coli GTP cyclohydrolase I (GCH1)having at least 94% sequence identity to native E. coli GCH1 having thesequence of SEQ ID NO:16 and comprising an amino acid substitutionselected from T1981, V179A, M99I, F214S and L200P, or a combination ofamino acid substitutions selected from (a) to (n): (a) I67V, T117I,A125D, H221R; (b) E62K, N170K, L215P; (c) V102M, L215P; (d) Q157L,H212R; (e) V28L, L215P, N222I; (f) T108N, I133F, E213K; (g) S5C, D57V,L215Q; (h) H29Y, I75V, V179M; (i) A14V, E46D, M61I, D97V; (j) V28A,G42D, E213K; (k) N52K, A68S, S207R; (l) A41G, K129N, I133F; (m) S3L,K184R, S199Y; and (n) H12R, N170D, G187S.
 15. A variant of E. coli GTPcyclohydrolase I (GCH1) having at least 98% sequence identity to nativeE. coli GCH1 having the sequence of SEQ ID NO:16, wherein the variantcomprises a mutation in amino acid residue T198, and wherein, in an E.coli cell comprising a PCD and at least one of a TPH, a TH and a PheH,the variant provides for an increased hydroxylation activity of at leastone of the TPH, TH and PheH as compared to native E. coli GCH1.
 16. Thevariant of claim 15, wherein the mutation is selected from T198I, T198S,T198V and T198L.
 17. The variant of claim 1, wherein the variant has atleast about 98% sequence identity to native E. coli GCH1 having thesequence of SEQ ID NO:16.
 18. The variant of claim 1, wherein thevariant provides for a hydroxylation activity of at least about 120% ascompared to native E. coli GCH1 having the sequence of SEQ ID NO:16. 19.The variant of claim 2, wherein the variant has at least about 98%sequence identity to native E. coli GCH1 having the sequence of SEQ IDNO:16.
 20. A variant of E. coli GCH1 consisting of SEQ ID NO:16 exceptfor a T198I mutation.
 21. The recombinant microbial cell of claim 10,wherein said Escherichia cell is an E. coli cell.